Novel antigen associated with the neovasculature of tumour metastases

ABSTRACT

The invention relates to a binding member that binds the Extra Domain-A (ED-A) isoform of fibronectin for the treatment of tumour metastases.

The present invention relates to detection and treatment of metastases,i.e. detection and treatment of secondary tumours arising at a site thatis distinct from a site of a primary tumour. The invention involves useof a binding member that binds the ED-A isoform of fibronectin,especially a binding member that binds domain ED-A of fibronectin.

The majority of cancer-related deaths are related to the metastaticspread of the disease (Hanahan and Weinberg 2000) and vigorousneovasculature is a characteristic feature of aggressive tumourmetastases.

Tumours are classified as either benign or malignant. Malignant tumoursare able to spread from the primary site (the primary tumour) to otherparts of the body while benign tumours cannot spread. Malignant tumourscan spread from their primary site by invasion and metastasis. Tumoursformed as a result of metastasis are known, for example, as metastases,secondary tumours, metastatic lesions or metastatic foci.

Angiogenesis describes the growth of new blood vessels from existingblood vessels. Tumours can induce angiogenesis through secretion ofvarious growth factors (e.g. Vascular Endothelial Growth Factor). Tumourangiogenesis allows tumours to grow beyond a few millimetres in diameterand is also a prerequisite for tumour metastasis. New blood vesselsformed as the result of angiogenesis form the neovasculature of thetumour or the tumour metastases.

Fibronectin (FN) is a glycoprotein and is widely expressed in a varietyof normal tissues and body fluids. It is a component of theextracellular matrix (ECM), and plays a role in many biologicalprocesses, including cellular adhesion, cellular migration, haemostasis,thrombosis, wound healing, tissue differentiation and oncogenictransformation.

Different FN isoforms are generated by alternative splicing of threeregions (ED-A, ED-B, IIICS) of the primary transcript FN pre-mRNA, aprocess that is modulated by cytokines and extracellular pH (Balza 1988;Carnemolla 1989; Borsi 1990; Borsi 1995). Fibronectin contains twotype-III globular extra-domains which may undergo alternative splicing:ED-A and ED-B (ffrench-Constant 1995, Hynes 1990, Kaspar et al. 2006).The ED-As of mouse fibronectin and human fibronectin are 96.7% identical(only 3 amino acids differ between the two 90 amino acid sequences, seeFIG. 5).

Expression of the ED-A of fibronectin has been reported in tumour cellsand in solid tumours at the mRNA level [see, e.g., (Jacobs et al. 2002,Matsumoto et al. 1999, Oyama et al. 1989, Tavian et al. 1994), at thelevel of isolated protein (Borsi et al. 1987) and at theimmunohistochemical level (Borsi et al. 1998, Heikinheimo et al. 1991,Koukoulis et al. 1993, Koukoulis et al. 1995, Lohi et al. 1995, Scarpinoet al. 1999). It has also been reported by Borsi et al., 1998, Exp CellRes, 240, 244-251, that ED-A is present in the neo-vasculature ofprimary tumours. However no indication that ED-A is associated with theneo-vasculature of tumour metastases has previously been made.

We show herein that the ED-A of fibronectin is selectively expressed inthe neovasculature of tumour metastases. As tumour blood vessels arereadily accessible for intravenously-administered therapeutic agents(Neri and Bicknell 2005, Rybak et al. 2006, Thorpe 2004, Trachsel andNeri 2006), binding molecules such as antibody molecules that bind theA-FN and/or the ED-A of fibronectin represent novel agents which may beused for the preparation of a medicament for the treatment of the tumourmetastases and/or tumour metastasis. The therapy of tumourneo-vasculature (tumour vascular targeting) is a promising approach forthe treatment of tumour metastases. Tumour vascular targeting aims atdisrupting the vasculature within the tumour itself, reducing blood flowto deprive the tumour of oxygen and nutrients, causing tumour celldeath.

Provided herein are anti-ED-A antibodies which selectively recognize thenew forming blood vessels of tumour metastases.

This invention in one aspect relates to the use of a binding member,e.g. an antibody molecule, that binds the Extra Domain-A (ED-A) isoformof fibronectin (A-FN), for the preparation of a medicament for thetreatment of a tumour metastases and/or tumour metastasis. In anotheraspect the invention relates to the use of a binding member, e.g. anantibody molecule, that binds the ED-A of fibronectin for thepreparation of a medicament for the treatment of tumour metastasesand/or tumour metastasis.

In a further aspect, the invention relates to the use of a bindingmember, e.g. an antibody molecule, that binds the ED-A isoform offibronectin for delivery, to the neovasculature of tumour metastases, ofa molecule conjugated to the binding member. In another aspect, theinvention relates to the use of a binding member, e.g. an antibodymolecule, that binds the ED-A of fibronectin for delivery, to theneovasculature of tumour metastases, of a molecule conjugated to thebinding member. In further aspects, the binding member may be used forthe manufacture of a medicament for delivery of such a molecule.

In a yet further aspect, the invention relates to the use of a bindingmember, e.g. an antibody molecule, that binds the ED-A isoform offibronectin for the manufacture of a diagnostic product for use indiagnosing a tumour metastases. In a yet further aspect, the inventionrelates to the use of a binding member, e.g. an antibody molecule, thatbinds the ED-A of fibronectin for the manufacture of a diagnosticproduct for use in diagnosing a tumour metastases.

The invention in one aspect also relates to a method of detecting ordiagnosing a tumour metastases in a human or animal comprising the stepsof:

(a) administering to the human or animal a binding member, e.g. anantibody molecule, which binds the ED-A isoform of fibronectin, and(b) determining the presence or absence of the binding member at a sitedistant from a site currently or previously occupied by a primary tumourin the human or animal body;wherein localisation of the binding member to a site distant from thesite currently or previously occupied by the primary tumour in the humanor animal indicates the presence of a tumour metastases.

The invention in another aspect relates to a method of detecting ordiagnosing a tumour metastases in a human or animal comprising the stepsof:

(a) administering to the human or animal a binding member, e.g. anantibody molecule, which binds the ED-A of fibronectin, and(b) determining the presence or absence of the binding member at a sitedistant from a site currently or previously occupied by a primary tumourin the human or animal body;wherein localisation of the binding member to a site distant from thesite currently or previously occupied by the primary tumour in the humanor animal indicates the presence of a tumour metastases.

The present invention also relates in one aspect to a method of treatinga tumour metastases in an individual comprising administering to theindividual a therapeutically effective amount of a medicament comprisinga binding member, e.g. an antibody molecule, which binds the ED-Aisoform of fibronectin. In another aspect, the present invention relatesto a method of treating a tumour metastases in an individual comprisingadministering to the individual a therapeutically effective amount of amedicament comprising a binding member, e.g. an antibody molecule, whichbinds the ED-A of fibronectin.

In another aspect, the invention relates to a method of delivering amolecule to the neovasculature of tumour metastases in a human or animalcomprising administering to the human or animal a binding member, e.g.an antibody molecule, which binds the ED-A isoform of fibronectin,wherein the binding member is conjugated to the molecule. In a furtheraspect, the invention relates to a method of delivering a molecule tothe neovasculature of tumour metastases in a human or animal comprisingadministering to the human or animal a binding member, e.g. an antibodymolecule which binds the ED-A of fibronectin, wherein the binding memberis conjugated to the molecule.

The binding member of the invention may be an antibody which binds theED-A isoform of fibronectin and/or the ED-A of fibronectin, comprisingone or more complementarity determining regions (CDRs) of antibody H1,B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9, or variants thereof.Preferably, a binding member of the invention is an antibody which bindsthe ED-A isoform of fibronectin and/or the ED-A of fibronectin,comprising one or more complementarity determining regions (CDRs) ofantibody B2, C5, D5, C8, F8, B7 or G9, or variants thereof. Mostpreferably, a binding member of the invention is an antibody which bindsthe ED-A isoform of fibronectin and/or the ED-A of fibronectin,comprising one or more complementarity determining regions (CDRs) ofantibody F8 or variants thereof.

The binding member of the invention may comprise a set of H and/or LCDRs of antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9, or a setof H and/or L CDRs of antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 orG9 with ten or fewer, e.g. one, two, three, four, or five, amino acidsubstitutions within the disclosed set of H and/or L CDRs. Preferably,the binding member of the invention comprises a set of H and/or L CDRsof antibody B2, C5, D5, C8, F8, B7 or G9 with ten or fewer, e.g. one,two, three, four, or five, amino acid substitutions within the disclosedset of H and/or L CDRs. Preferably, the binding member of the inventioncomprises a set of H and/or L CDRs of antibody F8 with ten or fewer,e.g. one, two, three, four, or five, amino acid substitutions within thedisclosed set of H and/or L CDRs.

Substitutions may potentially be made at any residue within the set ofCDRs, and may be within CDR1, CDR2 and/or CDR3.

For example, a binding member of the invention may comprise one or moreCDRs as described herein, e.g. a CDR3, and optionally also a CDR1 andCDR2 to form a set of CDRs.

A binding member of the invention may also comprise an antibodymolecule, e.g. a human antibody molecule. The binding member normallycomprises an antibody VH and/or VL domain. VH domains of binding membersare also provided as part of the invention. Within each of the VH and VLdomains are complementarity determining regions, (“CDRs”), and frameworkregions, (“FRs”). A VH domain comprises a set of HCDRs, and a VL domaincomprises a set of LCDRs. An antibody molecule may comprise an antibodyVH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It mayalternatively or also comprise an antibody VL domain comprising a VLCDR1, CDR2 and CDR3 and a framework. The VH and VL domains and CDRs ofantibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 are describedherein. All VH and VL sequences, CDR sequences, sets of CDRs and sets ofHCDRs and sets of LCDRs disclosed herein represent aspects andembodiments of a binding member for use in the invention. As describedherein, a “set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, a set ofHCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers toLCDR1, LCDR2 and LCDR3. Unless otherwise stated, a “set of CDRs”includes HCDRs and LCDRs.

A binding member of the invention may comprise an antibody VH domaincomprising complementarity determining regions HCDR1, HCDR2 and HCDR3and a framework, wherein HCDR1 is SEQ ID NO: 3, 23, 33, 43, 53, 63, 73,83, 93, 103 or 113, and wherein optionally HCDR2 is SEQ ID NO: 4 and/orHCDR3 is SEQ ID NO: 5. Preferably, the HCDR1 is SEQ ID NO: 23, 33, 43,53, 73, 83 or 103. Most preferably, the HCDR1 is SEQ ID NO: 83.

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although as discussed further below a VH or VLdomain alone may be used to bind antigen. Thus, a binding member of theinvention may further comprise an antibody VL domain comprisingcomplementarity determining regions LCDR1, LCDR2 and LCDR3 and aframework, wherein LCDR1 is SEQ ID NO: 6, 26, 36, 46, 56, 66, 76, 86,96, 106 or 116 and wherein optionally LCDR2 is SEQ ID NO: 7 and/or LCDR3is SEQ ID NO: 8. Preferably, the LCDR1 is SEQ ID NO: 26, 36, 46, 56, 76,86 or 106. Most preferably, the LCDR1 is SEQ ID NO: 86.

In one aspect the binding member of the invention is an isolatedantibody molecule for the ED-A of fibronectin, comprising a VH domainand a VL domain, wherein the VH domain comprises a framework and a setof complementarity determining regions HCDR1, HCDR2 and HCDR3 andwherein the VL domain comprises complementarity determining regionsLCDR1, LCDR2 and LCDR3 and a framework, and wherein

HCDR1 has amino acid sequence SEQ ID NO: 3, 23, 33, 43, 53, 63, 73, 83,93, 103 or 113,HCDR2 has amino acid sequence SEQ ID NO: 4,HCDR3 has amino acid sequence SEQ ID NO: 5,LCDR1 has amino acid sequence SEQ ID NO: 6, 26, 36, 46, 56, 66, 76, 86,96, 106 or 116;LCDR2 has amino acid sequence SEQ ID NO: 7; andLCDR3 has amino acid sequence SEQ ID NO: 8.

One or more CDRs or a set of CDRs of an antibody may be grafted into aframework (e.g. human framework) to provide an antibody molecule for usein the invention. Framework regions may comprise human germline genesegment sequences. Thus, the framework may be germlined, whereby one ormore residues within the framework are changed to match the residues atthe equivalent position in the most similar human germline framework. Abinding member of the invention may be an isolated antibody moleculehaving a VH domain comprising a set of HCDRs in a human germlineframework, e.g. DP47. Normally the binding member also has a VL domaincomprising a set of LCDRs, e.g. in a human germline framework. The humangermline framework of the VL domain may be DPK22.

A VH domain of the invention may have amino acid sequence SEQ ID NO: 1,21, 31, 41, 51, 61, 71, 81, 91, 101 or 111. Preferably, a VH domain ofthe invention has amino acid sequence SEQ ID NO: 21, 31, 41, 51, 71, 81or 101. Most preferably, a VH domain of the invention has amino acidsequence SEQ ID NO: 81. A VL domain of the invention may have the aminoacid SEQ ID NO: 2, 22, 32, 42, 52, 62, 72, 82, 92, 102 or 112.Preferably, a VL domain of the invention has amino acid SEQ ID NO: 22,32, 42, 52, 72, 82 or 102. Most preferably, a VL domain of the inventionhas amino acid SEQ ID NO: 82.

A binding member of the invention may be a single chain Fv (scFv),comprising a VH domain and a VL domain joined via a peptide linker. Theskilled person may select an appropriate length and sequence of linker,e.g. at least 5 or 10 amino acids in length, up to about 15, 20 or 25amino acids in length. The scFv may consist of or comprise amino acidsequence SEQ ID NO: 9.

A binding member of the invention may be a diabody (WO94/13804; Holliger1993a), which is a molecule comprising a first polypeptide with a VHdomain and a VL domain joined via a peptide linker and a secondpolypeptide with a VH domain and a VL domain joined via a peptide linkerwherein the VH domain and the VL domain of the first polypeptide pairwith the VL domain and VH domain of the second polypeptide,respectively. The first and second polypeptides may be the same (wherebypairing results in a bivalent molecule) or different (whereby pairingresults in a bispecific molecule). The skilled person may select anappropriate length and sequence of linker, e.g. 5 or fewer amino acidsin length. The linker may have amino acid sequence SEQ ID NO: 28.

A binding member of the invention may comprise an antigen-binding sitewithin a non-antibody molecule, normally provided by one or more CDRse.g. a set of CDRs in a non-antibody protein scaffold. Binding members,including non-antibody and antibody molecules, are described in moredetail elsewhere herein.

A binding member of the invention may be conjugated to a molecule thathas biocidal or cytotoxic activity. Alternatively, a binding member ofthe invention may be conjugated to a radioisotope. As a furtheralterative, a binding member of the invention may be labelled with adetectable label.

These and other aspects of the invention are described in further detailbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the perfusion based proteomicmethodology used for the comparative analysis of accessible proteins inliver from healthy mice and F9 liver metastases from mice. FIG. 1B showsthe large metastatic foci developed by F9 liver metastases. FIG. 1Cshows the selective and efficient staining of the blood vessels of F9liver metastases (Metastasis) as well as the strong staining of theblood vessels and labelling of some sinusoids in normal liver (Liver).Staining corresponds to darker lines and it is obtained aftertumour-bearing mice were perfused with 15 ml of a 1.8 mM solution ofsulfosuccinimidyl-6-[biotin-amido]hexanoate (1 mg/ml) in PBS, pH 7.4,supplemented with 10% Dextran-40 as plasma expander under terminalanaesthesia followed by histochemical staining with astreptavidin-alkaline phosphatase conjugate.

FIG. 2A is a schematic showing the location of the fibronectin peptidesidentified in the proteomic analysis of normal mouse liver (Normal) andF9 liver metastases from mice (Tumor) on the fibronectin domainstructure (SEQ ID NO: 117). FIG. 2B shows the peptides identified in theproteomic analysis of normal mouse liver samples and F9 liver metastasesfrom mice were submitted to an LC-MS/MS experiment. The peptides werefirst separated by HPLC and subsequently eluted in 192 fractions. Eachfraction was spotted as a separate spot onto a MALDI target plate andMALDI TOF MS spectra were acquired of each fraction. Mass spectra of twodifferent particular HPLC fractions (upper row and middle row,respectively) are shown for three replicate F9 mouse liver metastasessamples (see panel labelled: “Liver metastasis”) and three replicatesamples of normal mouse liver (see panel labelled: “Normal liver”). Theion peak heights are normalized to the internal standard (see Materialsand Methods) and thus allow a semi-quantitative comparison ofcorresponding peptides in the different samples. In the upper row, thepeak indicated with an arrow (labelled “FN” in the first sample shown)corresponds to the peptide FLTTTPNSLLVSWQAPR (SEQ ID NO: 15) whichderives from a constant region of fibronectin (fibronectin-type-IIIdomain 16). The ion peak of this peptide is higher in the F9 mouse livermetastases samples (Liver metastasis) but is also present in the normalmouse liver samples (Normal liver), indicating that the fibronectinmolecule is, in principle, present in both F9 mouse liver metastases andnormal mouse liver but it seems to be more abundant in the F9 mouseliver metastases samples. In the middle row, the peak indicated with theright hand arrow (labelled “EDA”) corresponds to the peptideIAWESPQGQVSR (SEQ ID NO: 16) which derives from the alternativelyspliced extra-domain A of fibronectin. This ED-A peptide is onlydetectable in F9 mouse liver metastases samples (Liver metastasis) andnot in the normal mouse liver samples (Normal liver). The referencepeptide indicated with the left hand arrow (labelled “ref”) was used toidentify the HPLC fraction in which the ED-A peptide elutes. This meansthat the presence of the peak of the reference peptide in the spectrashown for the normal mouse liver samples (Normal liver) is proof thatthe mass spectra of the fractions in which the ED-A peptide would bedetectable, if it was present in the normal mouse liver samples, isshown. The bottom row shows a close-up view of the mass spectra at theposition of the ED-A peptide ion peak (indicated by the arrow) provingthe absence of this peptide from the normal liver samples.

FIG. 3A shows immunohistochemical staining (darker lines) of F9 livermetastases and adjacent normal mouse liver tissue with flag-taggedparent anti-ED-A antibody (anti-ED-A) revealed a strong vascular patternof staining in the metastases, while no specific staining was detectablein adjacent normal liver tissue. In the negative controls (Control) theflag-tagged parent anti-ED-A antibody was omitted. The staining patternobserved with the flag-tagged parent anti-ED-A antibody is similar tothe staining pattern observed with flag-tagged anti-ED-B scFv(L19)antibody (anti-EDB) which recognizes the fibronectin extra-domain B, awell established marker of neovascular structures. FIG. 3B shows theorgans (spleen, heart, lung and a liver portion with two metastases) ofSv190 mice which were injected with F9DR tumour cells, and three weekslater were further injected in the tail vein with (200 μl/mouse, i.e. 60μg antibody/mouse) Alexa 750-labelled parent anti-ED-A antibody (in afinal concentration of 0.3 mg/ml). The mouse organs were excised sixhours after injection of the Alexa 750-labelled parent anti-ED-Aantibody. Alexa 750-labelled parent anti-ED-A antibody staining wasvisualized using a home-built infrared fluorescent imager (Birchler etal. 1999) equipped with a tungsten halogen lamp, excitation and emissionfilters specific for Alexa 750, and a monochrome CCD camera.

FIG. 4: Shows ED-A expression in human metastases. The flag-taggedparent anti-ED-A antibody was used to assess the expression of ED-A inhuman metastases by immunohistochemistry. While no positive staining wasdetectable in negative controls (Control) omitting the flag-taggedparent anti-ED-A antibody and only a very weak background staining wasobserved on human normal lung tissue sections (Normal human lung) withthe flag-tagged parent anti-ED-A antibody, human pulmonary metastases(Human pulmonary metastasis of RCC [renal cell carcinoma]) were stronglypositively stained with the flag-tagged parent anti-ED-A antibody(anti-EDA) as shown by the darker lines and shades. The staining patternof the flag-tagged parent anti-ED-A antibody is mainly vascular and issimilar to the staining pattern observed with the flag-tagged anti-ED-BscFv(L19) antibody (anti-EDB) which recognizes the fibronectinextra-domain B, a well established marker of neovascular structures.Similar results were obtained by immunohistochemical analysis of humanliver metastases of colorectal carcinoma (Human liver metastasis of CRC)with the flag-tagged parent anti-ED-A antibody. The flag-tagged parentanti-ED-A antibody reveals a strong vascular and stromal stainingpattern human liver metastases of colorectal carcinoma.

FIG. 5: Shows an alignment between the human ED-A (top sequence; SEQ IDNO: 118) and the mouse ED-A (bottom sequence; SEQ ID NO: 119). Theasterisks indicate the amino acid positions where the amino acids of thehuman ED-A and the mouse ED-A are identical.

FIG. 6A shows the nucleotide sequence of the anti-ED-A antibody H1 heavychain (VH) (SEQ ID NO: 12). The nucleotide sequence of the heavy chainCDR1 of anti-ED-A antibody H1 is underlined. The nucleotide sequence ofthe heavy chain CDR2 of the anti-ED-A antibody H1 is shown in italicsand underlined. The nucleotide sequence of the heavy chain CDR3 ofanti-ED-A antibody H1 is shown in bold and underlined. FIG. 6B shows thenucleotide sequence of the anti-ED-A antibody H1 linker sequence (SEQ IDNO: 14). FIG. 6C shows the nucleotide sequence of the anti-ED-A antibodyH1 light chain (VL) (SEQ ID NO: 13). The nucleotide sequence of thelight chain CDR1 of anti-ED-A antibody H1 is underlined. The nucleotidesequence of the light chain CDR2 of the anti-ED-A antibody H1 is shownin italics and underlined. The nucleotide sequence of the light chainCDR3 of anti-ED-A antibody H1 is shown in bold and underlined.

FIG. 7A shows the amino acid sequence of the anti-ED-A antibody H1 heavychain (VH) (SEQ ID NO: 1). The amino acid sequence of the heavy chainCDR1 (SEQ ID NO: 3) of anti-ED-A antibody H1 is underlined. The aminoacid sequence of the heavy chain CDR2 (SEQ ID NO: 4) of the anti-ED-Aantibody H1 is shown in italics and underlined. The amino acid sequenceof the heavy chain CDR3 (SEQ ID NO: 5) of anti-ED-A antibody H1 is shownin bold and underlined. FIG. 7B shows the amino acid sequence of theanti-ED-A antibody H1 linker sequence (SEQ ID NO: 11). FIG. 7C shows theamino acid sequence of the anti-ED-A antibody H1 light chain (VL) (SEQID NO: 2). The amino acid sequence of the light chain CDR1 (SEQ ID NO:6) of anti-ED-A antibody H1 is underlined. The amino acid sequence ofthe light chain CDR2 (SEQ ID NO: 7) of the anti-ED-A antibody H1 isshown in italics and underlined. The amino acid sequence of the lightchain CDR3 (SEQ ID NO: 8) of anti-ED-A antibody H1 is shown in bold andunderlined.

FIG. 8: Shows the biodistribution of the F8 diabody in F9 tumour bearingmice. Four F9 tumour bearing mice were injected intravenously with I¹²⁵labelled F8 diabody. The mice were sacrificed after twenty four hoursand tumour, liver, lung, spleen, heart, kidney, intestine, tail andblood removed. The tumour, liver, lung, spleen, heart, kidney,intestine, tail and blood were then radioactively counted. Thepercentage (%) of the injected dose (ID) of I¹²⁵ labelled F8 diabodydetected per gram (g) of tumour, liver, lung, spleen, heart, kidney,intestine, tail and blood is shown in FIG. 8. The F9 tumours (tumours)contained about four times more of the ID than any of the other mousetissues analyzed.

TERMINOLOGY Fibronectin

Fibronectin is an antigen subject to alternative splicing, and a numberof alternative isoforms of fibronectin are known, as described elsewhereherein. Extra Domain-A (EDA or ED-A) is also known as ED, extra type IIIrepeat A (EIIIA) or EDI. The sequence of human ED-A has been publishedby Kornblihtt et al. (1984), Nucleic Acids Res. 12, 5853-5868 andPaolella et al. (1988), Nucleic Acids Res. 16, 3545-3557. The sequenceof human ED-A is also available on the SwissProt database as amino acids1631-1720 (Fibronectin type-III 12; extra domain 2) of the amino acidsequence deposited under accession number P02751. The sequence of mouseED-A is available on the SwissProt database as amino acids 1721-1810(Fibronectin type-III 13; extra domain 2) of the amino acid sequencedeposited under accession number P11276.

The ED-A isoform of fibronectin (A-FN) contains the Extra Domain-A(ED-A). The sequence of the human A-FN can be deduced from thecorresponding human fibronectin precursor sequence which is available onthe SwissProt database under accession number P02751. The sequence ofthe mouse A-FN can be deduced from the corresponding mouse fibronectinprecursor sequence which is available on the SwissProt database underaccession number P11276. The A-FH may be the human ED-A isoform offibronectin. The ED-A may be the Extra Domain-A of human fibronectin.

ED-A is a 90 amino acid sequence which is inserted into fibronectin (FN)by alternative splicing and is located between domain 11 and 12 of FN(Borsi et al., 1987, J. Cell Biol., 104, 595-600). ED-A is mainly absentin the plasma form of FN but is abundant during embryogenesis, tissueremodelling, fibrosis, cardiac transplantation and solid tumour growth.

Alternative Splicing

Alternative splicing refers to the occurrence of different patterns ofsplicing of a primary RNA transcript of DNA to produce different mRNAs.After excision of introns, selection may determine which exons arespliced together to form the mRNA. Alternative splicing leads toproduction of different isoforms containing different exons and/ordifferent numbers of exons. For example one isoform may comprise anadditional amino acid sequence corresponding to one or more exons, whichmay comprise one or more domains.

Binding Member

This describes one member of a pair of molecules that bind one another.The members of a binding pair may be naturally derived or wholly orpartially synthetically produced. One member of the pair of moleculeshas an area on its surface, or a cavity, which binds to and is thereforecomplementary to a particular spatial and polar organization of theother member of the pair of molecules. Examples of types of bindingpairs are antigen-antibody, biotin-avidin, hormone-hormone receptor,receptor-ligand, enzyme-substrate. The present invention is concernedwith antigen-antibody type reactions.

A binding member normally comprises a molecule having an antigen-bindingsite. For example, a binding member may be an antibody molecule or anon-antibody protein that comprises an antigen-binding site.

An antigen binding site may be provided by means of arrangement ofcomplementarity determining regions (CDRs) on non-antibody proteinscaffolds such as fibronectin or cytochrome B etc. (Haan & Maggos, 2004;Koide 1998; Nygren 1997), or by randomising or mutating amino acidresidues of a loop within a protein scaffold to confer bindingspecificity for a desired target. Scaffolds for engineering novelbinding sites in proteins have been reviewed in detail by Nygren et al.(1997). Protein scaffolds for antibody mimics are disclosed inWO/0034784, which is herein incorporated by reference in its entirety,in which the inventors describe proteins (antibody mimics) that includea fibronectin type III domain having at least one randomised loop. Asuitable scaffold into which to graft one or more CDRs, e.g. a set ofHCDRs, may be provided by any domain member of the immunoglobulin genesuperfamily. The scaffold may be a human or non-human protein. Anadvantage of a non-antibody protein scaffold is that it may provide anantigen-binding site in a scaffold molecule that is smaller and/oreasier to manufacture than at least some antibody molecules. Small sizeof a binding member may confer useful physiological properties such asan ability to enter cells, penetrate deep into tissues or reach targetswithin other structures, or to bind within protein cavities of thetarget antigen. Use of antigen binding sites in non-antibody proteinscaffolds is reviewed in Wess, 2004. Typical are proteins having astable backbone and one or more variable loops, in which the amino acidsequence of the loop or loops is specifically or randomly mutated tocreate an antigen-binding site that binds the target antigen. Suchproteins include the IgG-binding domains of protein A from S. aureus,transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type IIIdomain) and lipocalins. Other approaches include synthetic “Microbodies”(Selecore GmbH), which are based on cyclotides—small proteins havingintra-molecular disulphide bonds.

In addition to antibody sequences and/or an antigen-binding site, abinding member according to the present invention may comprise otheramino acids, e.g. forming a peptide or polypeptide, such as a foldeddomain, or to impart to the molecule another functional characteristicin addition to ability to bind antigen. Binding members of the inventionmay carry a detectable label, or may be conjugated to a toxin or atargeting moiety or enzyme (e.g. via a peptidyl bond or linker). Forexample, a binding member may comprise a catalytic site (e.g. in anenzyme domain) as well as an antigen binding site, wherein the antigenbinding site binds to the antigen and thus targets the catalytic site tothe antigen. The catalytic site may inhibit biological function of theantigen, e.g. by cleavage.

Although, as noted, CDRs can be carried by non-antibody scaffolds, thestructure for carrying a CDR or a set of CDRs of the invention willgenerally be an antibody heavy or light chain sequence or substantialportion thereof in which the CDR or set of CDRs is located at a locationcorresponding to the CDR or set of CDRs of naturally occurring VH and VLantibody variable domains encoded by rearranged immunoglobulin genes.The structures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat 1987, and updates thereof, nowavailable on the Internet (at immuno.bme.nwu.edu or find “Kabat” usingany search engine).

By CDR region or CDR, it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulin as definedby Kabat et al. (1987), (Kabat 1991a, and later editions). An antibodytypically contains 3 heavy chain CDRs and 3 light chain CDRs. The termCDR or CDRs is used here in order to indicate, according to the case,one of these regions or several, or even the whole, of these regionswhich contain the majority of the amino acid residues responsible forthe binding by affinity of the antibody for the antigen or the epitopewhich it recognizes.

Among the six short CDR sequences, the third CDR of the heavy chain(HCDR3) has a greater size variability (greater diversity essentiallydue to the mechanisms of arrangement of the genes which give rise toit). It can be as short as 2 amino acids although the longest size knownis 26. Functionally, HCDR3 μlays a role in part in the determination ofthe specificity of the antibody (Segal 1974; Amit 1986; Chothia 1987;Chothia 1989; Caton 1990; Sharon 1990a; Sharon 1990b; Rabat et al.,1991b).

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteincomprising an antibody antigen-binding site. It must be understood herethat the invention does not relate to the antibodies in natural form,that is to say they are not in their natural environment but that theyhave been able to be isolated or obtained by purification from naturalsources, or else obtained by genetic recombination, or by chemicalsynthesis, and that they can then contain unnatural amino acids as willbe described later. Antibody fragments that comprise an antibodyantigen-binding site include, but are not limited to, antibody moleculessuch as Fab, Fab′, Fab′-SH, scFv, Fv, dAb, Fd; and diabodies.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules that bind the target antigen. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe CDRs, of an antibody to the constant regions, or constant regionsplus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400, and a large body ofsubsequent literature. A hybridoma or other cell producing an antibodymay be subject to genetic mutation or other changes, which may or maynot alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any binding member orsubstance having an antibody antigen-binding site with the requiredspecificity and/or binding to antigen. Thus, this term covers antibodyfragments and derivatives, including any polypeptide comprising anantibody antigen-binding site, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an antibody antigen-bindingsite, or equivalent, fused to another polypeptide (e.g. derived fromanother species or belonging to another antibody class or subclass) aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023, and a large body ofsubsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann & Dubel (2001).Phage display, another established technique for generating bindingmembers has been described in detail in many publications such asWO92/01047 (discussed further below) and U.S. Pat. No. 5,969,108, U.S.Pat. No. 5,565,332, U.S. Pat. No. 5,733,743, U.S. Pat. No. 5,858,657,U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,872,215, U.S. Pat. No.5,885,793, U.S. Pat. No. 5,962,255, U.S. Pat. No. 6,140,471, U.S. Pat.No. 6,172,197, U.S. Pat. No. 6,225,447, U.S. Pat. No. 6,291,650, U.S.Pat. No. 6,492,160, U.S. Pat. No. 6,521,404 and Kontermann & Dubel(2001). Transgenic mice in which the mouse antibody genes areinactivated and functionally replaced with human antibody genes whileleaving intact other components of the mouse immune system, can be usedfor isolating human antibodies (Mendez 1997).

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.(2000) or Krebs et al. (2001).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward 1989; McCafferty 1990; Holt 2003), which consists of a VHor a VL domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird 1988; Huston 1988); (viii) bispecific singlechain Fv dimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; Holliger1993a). Fv, scFv or diabody molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter 1996). Minibodies comprising a scFv joined to a CH3 domain mayalso be made (Hu 1996). Other examples of binding fragments are Fab′,which differs from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH1 domain, including one ormore cysteines from the antibody hinge region, and Fab′-SH, which is aFab′ fragment in which the cysteine residue(s) of the constant domainsbear a free thiol group.

Antibody fragments of the invention can be obtained starting from any ofthe antibody molecules described herein, e.g. antibody moleculescomprising VH and/or VL domains or CDRs of any of antibodies describedherein, by methods such as digestion by enzymes, such as pepsin orpapain and/or by cleavage of the disulfide bridges by chemicalreduction. In another manner, antibody fragments of the presentinvention may be obtained by techniques of genetic recombinationlikewise well known to the person skilled in the art or else by peptidesynthesis by means of, for example, automatic peptide synthesizers suchas those supplied by the company Applied Biosystems, etc., or by nucleicacid synthesis and expression.

Functional antibody fragments according to the present invention includeany functional fragment whose half-life is increased by a chemicalmodification, especially by PEGylation, or by incorporation in aliposome.

A dAb (domain antibody) is a small monomeric antigen-binding fragment ofan antibody, namely the variable region of an antibody heavy or lightchain (Holt 2003). VH dAbs occur naturally in camelids (e.g. camel,llama) and may be produced by immunizing a camelid with a targetantigen, isolating antigen-specific B cells and directly cloning dAbgenes from individual B cells. dAbs are also producible in cell culture.Their small size, good solubility and temperature stability makes themparticularly physiologically useful and suitable for selection andaffinity maturation. A binding member of the present invention may be adAb comprising a VH or VL domain substantially as set out herein, or aVH or VL domain comprising a set of CDRs substantially as set outherein.

As used herein, the phrase “substantially as set out” refers to thecharacteristic(s) of the relevant CDRs of the VH or VL domain of bindingmembers described herein will be either identical or highly similar tothe specified regions of which the sequence is set out herein. Asdescribed herein, the phrase “highly similar” with respect to specifiedregion(s) of one or more variable domains, it is contemplated that from1 to about 5, e.g. from 1 to 4, including 1 to 3, or 1 or 2, or 3 or 4,amino acid substitutions may be made in the CDR and/or VH or VL domain.

Bispecific or bifunctional antibodies form a second generation ofmonoclonal antibodies in which two different variable regions arecombined in the same molecule (Holliger 1999). Their use has beendemonstrated both in the diagnostic field and in the therapy field fromtheir capacity to recruit new effector functions or to target severalmolecules on the surface of tumor cells. Where bispecific antibodies areto be used, these may be conventional bispecific antibodies, which canbe manufactured in a variety of ways (Holliger 1993b), e.g. preparedchemically or from hybrid hybridomas, or may be any of the bispecificantibody fragments mentioned above. These antibodies can be obtained bychemical methods (Glennie 1987; Repp 1995) or somatic methods (Staerz1986; Suresh 1986) but likewise by genetic engineering techniques whichallow the heterodimerization to be forced and thus facilitate theprocess of purification of the antibody sought (Merchand 1998). Examplesof bispecific antibodies include those of the BiTE™ technology in whichthe binding domains of two antibodies with different specificity can beused and directly linked via short flexible peptides. This combines twoantibodies on a short single polypeptide chain, Diabodies and scFv canbe constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific antibodies can be constructed as entire IgG, as bispecificFab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against a target antigen, then a library can be made where theother arm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by alternative engineeringmethods as described in Ridgeway 1996.

Various methods are available in the art for obtaining antibodiesagainst a target antigen. The antibodies may be monoclonal antibodies,especially of human, murine, chimeric or humanized origin, which can beobtained according to the standard methods well known to the personskilled in the art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane 1988) or to the technique of preparationfrom hybridomas described by Kohler and Milstein, 1975.

Monoclonal antibodies can be obtained, for example, from an animal cellimmunized against A-FN, or one of its fragments containing the epitoperecognized by said monoclonal antibodies, e.g. a fragment comprising orconsisting of ED-A, or a peptide fragment of ED-A. The A-FIT, or one ofits fragments, can especially be produced according to the usual workingmethods, by genetic recombination starting with a nucleic acid sequencecontained in the cDNA sequence coding for A-FIT or fragment thereof, bypeptide synthesis starting from a sequence of amino acids comprised inthe peptide sequence of the A-FN and/or fragment thereof.

Monoclonal antibodies can, for example, be purified on an affinitycolumn on which A-FN or one of its fragments containing the epitoperecognized by said monoclonal antibodies, e.g. a fragment comprising orconsisting of ED-A or a peptide fragment of ED-A, has previously beenimmobilized. Monoclonal antibodies can be purified by chromatography onprotein A and/or G, followed or not followed by ion-exchangechromatography aimed at eliminating the residual protein contaminants aswell as the DNA and the LPS, in itself, followed or not followed byexclusion chromatography on Sepharose gel in order to eliminate thepotential aggregates due to the presence of dimers or of othermultimers. The whole of these techniques may be used simultaneously orsuccessively.

Antigen-Binding Site

This describes the part of a molecule that binds to and is complementaryto all or part of the target antigen. In an antibody molecule it isreferred to as the antibody antigen-binding site, and comprises the partof the antibody that binds to and is complementary to all or part of thetarget antigen. Where an antigen is large, an antibody may only bind toa particular part of the antigen, which part is termed an epitope. Anantibody antigen-binding site may be provided by one or more antibodyvariable domains. An antibody antigen-binding site may comprise anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

Isolated

This refers to the state in which binding members of the invention ornucleic acid encoding such binding members, will generally be inaccordance with the present invention. Thus, binding members, VH and/orVL domains of the present invention may be provided isolated and/orpurified, e.g. from their natural environment, in substantially pure orhomogeneous form, or, in the case of nucleic acid, free or substantiallyfree of nucleic acid or genes of origin other than the sequence encodinga polypeptide with the required function. Isolated members and isolatednucleic acid will be free or substantially free of material with whichthey are naturally associated such as other polypeptides or nucleicacids with which they are found in their natural environment, or theenvironment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practised in vitro or invivo. Members and nucleic acid may be formulated with diluents oradjuvants and still for practical purposes be isolated—for example themembers will normally be mixed with gelatin or other carriers if used tocoat microtitre plates for use in immunoassays, or will be mixed withpharmaceutically acceptable carriers or diluents when used in diagnosisor therapy. Binding members may be glycosylated, either naturally or bysystems of heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC85110503) cells, or they may be (for example if produced by expressionin a prokaryotic cell) unglycosylated.

Heterogeneous preparations comprising antibody molecules also form partof the invention. For example, such preparations may be mixtures ofantibodies with full-length heavy chains and heavy chains lacking theC-terminal lysine, with various degrees of glycosylation and/or withderivatized amino acids, such as cyclization of an N-terminal glutamicacid to form a pyroglutamic acid residue.

One or more binding members for an antigen, e.g. the A-FN or the ED-A offibronectin, may be obtained by bringing into contact a library ofbinding members according to the invention and the antigen or a fragmentthereof, e.g. a fragment comprising or consisting of ED-A or a peptidefragment of ED-A and selecting one or more binding members of thelibrary able to bind the antigen.

An antibody library may be screened using Iterative Colony FilterScreening (ICES). In ICFS, bacteria containing the DNA encoding severalbinding specificities are grown in a liquid medium and, once the stageof exponential growth has been reached, some billions of them aredistributed onto a growth support consisting of a suitably pre-treatedmembrane filter which is incubated until completely confluent bacteriaecolonies appear. A second trap substrate consists of another membranefilter, pre-humidified and covered with the desired antigen.

The trap membrane filter is then placed onto a plate containing asuitable culture medium and covered with the growth filter with thesurface covered with bacterial colonies pointing upwards. The sandwichthus obtained is incubated at room temperature for about 16 h. It isthus possible to obtain the expression of the genes encoding antibodyfragments scFv having a spreading action, so that those fragmentsbinding specifically with the antigen which is present on the trapmembrane are trapped. The trap membrane is then treated to point outbound antibody fragments scFv with colorimetric techniques commonly usedto this purpose.

The position of the coloured spots on the trap filter allows to go backto the corresponding bacterial colonies which are present on the growthmembrane and produced the antibody fragments trapped. Such colonies aregathered and grown and the bacteria—a few millions of them aredistributed onto a new culture membrane repeating the proceduresdescribed above. Analogous cycles are then carried out until thepositive signals on the trap membrane correspond to single positivecolonies, each of which represents a potential source of monoclonalantibody fragments directed against the antigen used in the selection.ICFS is described in e.g. WO0246455, which is incorporated herein byreference.

A library may also be displayed on particles or molecular complexes,e.g. replicable genetic packages such bacteriophage (e.g. T7) particles,or other in vitro display systems, each particle or molecular complexcontaining nucleic acid encoding the antibody VH variable domaindisplayed on it, and optionally also a displayed VL domain if present.Phage display is described in WO92/01047 and e.g. U.S. Pat. No.5,969,108, U.S. Pat. No. 5,565,332, U.S. Pat. No. 5,733,743, U.S. Pat.No. 5,858,657, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,872,215, U.S.Pat. No. 5,885,793, U.S. Pat. No. 5,962,255, U.S. Pat. No. 6,140,471,U.S. Pat. No. 6,172,197, U.S. Pat. No. 6,225,447, U.S. Pat. No.6,291,650, U.S. Pat. No. 6,492,160 and U.S. Pat. No. 6,521,404, each ofwhich is herein incorporated by reference in its entirety.

Following selection of binding members able to bind the antigen anddisplayed on bacteriophage or other library particles or molecularcomplexes, nucleic acid may be taken from a bacteriophage or otherparticle or molecular complex displaying a said selected binding member.Such nucleic acid may be used in subsequent production of a bindingmember or an antibody VH or VL variable domain by expression fromnucleic acid with the sequence of nucleic acid taken from abacteriophage or other particle or molecular complex displaying a saidselected binding member.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected binding member may beprovided in isolated form, as may a binding member comprising such a VHdomain.

Ability to bind the A-FN or the ED-A of fibronectin or other targetantigen or isoform may be further tested, e.g. ability to compete withe.g. any one of anti-ED-A antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7,E8 or G9 for binding to the A-FN or a fragment of the A-FN, e.g. theED-A of fibronectin.

A binding member of the invention may bind the A-FN and/or the ED-A offibronectin specifically. A binding member of the present invention maybind the A-FN and/or the ED-A of fibronectin with the same affinity asanti-ED-A antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9, e.g. inscFv format, or with an affinity that is better. A binding member of theinvention may bind the A-FN and/or the ED-A of fibronectin with a K_(D)of 3×10⁻⁸ M or an affinity that is better. Preferably, a binding memberof the invention binds the A-FN and/or the ED-A of fibronectin with aK_(D) of 2×10⁻⁸ M or an affinity that is better. More preferably, abinding member of the invention binds the A-FN and/or the ED-A offibronectin with a K_(D) of 1.7×10⁻⁸ M or an affinity that is better.Yet more preferably, a binding member of the invention binds the A-FNand/or the ED-A of fibronectin with a K_(D) of 1.4×10⁻⁸ M or an affinitythat is better. Most preferably, a binding member of the invention bindsthe A-FH and/or the ED-A of fibronectin with a K_(D) of 3×10⁻⁹ M or anaffinity that is better.

A binding member of the present invention may bind to the same epitopeon A-FH and/or the ED-A of fibronectin as anti-ED-A antibody H1, B2, C5,D5, E5, C8, F8, F1, B7, E8 or G9.

A binding member of the invention may not show any significant bindingto molecules other than the A-FN and/or the ED-A of fibronectin. Inparticular the binding member may not bind other isoforms offibronectin, for example the ED-B isoform and/or the IIICS isoform offibronectin.

Variants of antibody molecules disclosed herein may be produced and usedin the present invention. The techniques required to make substitutionswithin amino acid sequences of CDRs, antibody VH or VL domains andbinding members generally are available in the art. Variant sequencesmay be made, with substitutions that may or may not be predicted to havea minimal or beneficial effect on activity, and tested for ability tobind A-FN and/or the ED-A of fibronectin and/or for any other desiredproperty.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), may be less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, may be 5, 4, 3, 2 or 1. Alterations may be made in one ormore framework regions and/or one or more CDRs. The alterations normallydo not result in loss of function, so a binding member comprising athus-altered amino acid sequence may retain an ability to bind A-FNand/or the ED-A of fibronectin. For example, it may retain the samequantitative binding as a binding member in which the alteration is notmade, e.g. as measured in an assay described herein. The binding membercomprising a thus-altered amino acid sequence may have an improvedability to bind A-FN and/or the ED-A of fibronectin.

Novel VH or VL regions carrying CDR-derived sequences of the inventionmay be generated using random mutagenesis of one or more selected VHand/or VL genes to generate mutations within the entire variable domain.In some embodiments one or two amino acid substitutions are made withinan entire variable domain or set of CDRs. Another method that may beused is to direct mutagenesis to CDR regions of VH or VL genes.

As noted above, a CDR amino acid sequence substantially as set outherein may be carried as a CDR in a human antibody variable domain or asubstantial portion thereof. The HCDR3 sequences substantially as setout herein represent embodiments of the present invention and forexample each of these may be carried as a HCDR3 in a human heavy chainvariable domain or a substantial portion thereof.

Variable domains employed in the invention may be obtained or derivedfrom any germ-line or rearranged human variable domain, or may be asynthetic variable domain based on consensus or actual sequences ofknown human variable domains. A variable domain can be derived from anon-human antibody. A CDR sequence of the invention (e.g. CDR3) may beintroduced into a repertoire of variable domains lacking a CDR (e.g.CDR3), using recombinant DNA technology. For example, Marks et al.(1992) describe methods of producing repertoires of antibody variabledomains in which consensus primers directed at or adjacent to the 5′ endof the variable domain area are used in conjunction with consensusprimers to the third framework region of human VH genes to provide arepertoire of VH variable domains lacking a CDR3. Marks at al. furtherdescribe how this repertoire may be combined with a CDR3 of a particularantibody. Using analogous techniques, the CDR3-derived sequences of thepresent invention may be shuffled with repertoires of VH or VL domainslacking a CDR3, and the shuffled complete VH or VL domains combined witha cognate VL or VH domain to provide binding members of the invention.The repertoire may then be displayed in a suitable host system such asthe phage display system of WO92/01047, which is herein incorporated byreference in its entirety, or any of a subsequent large body ofliterature, including Kay, Winter & McCafferty (1996), so that suitablebinding members may be selected. A repertoire may consist of fromanything from 10⁴ individual members upwards, for example at least 10⁵,at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹ or at least 10¹⁰members.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains that are then screened for a bindingmember or binding members for the A-FN and/or the ED-A of fibronectin.

One or more of the HCDR1, HCDR2 and HCDR3 of antibody H1, B2, C5, D5,E5, C8, F8, F1, B7, E8 or G9, or the set of HCDRs may be employed,and/or one or more of the X LCDR1, LCDR2 and LCDR3 of antibody H1, B2,C5, D5, E5, C8, F8, F1, B7, E8 or G9 or the set of LCDRs of antibody H1,B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9 may be employed.

Similarly, other VH and VL domains, sets of CDRs and sets of HCDRsand/or sets of LCDRs disclosed herein may be employed.

The A-FN and/or the ED-A of fibronectin may be used in a screen forbinding members, e.g. antibody molecules, for use in the preparation ofa medicament for the treatment of tumour metastases. The screen may ascreen of a repertoire as disclosed elsewhere herein.

In some embodiments, a substantial portion of an immunoglobulin variabledomain will comprise at least the three CDR regions, together with theirintervening framework regions. The portion may also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of binding members ofthe present invention made by recombinant DNA techniques may result inthe introduction of N- or C-terminal residues encoded by linkersintroduced to facilitate cloning or other manipulation steps. Othermanipulation steps include the introduction of linkers to join variabledomains of the invention to further protein sequences including antibodyconstant regions, other variable domains (for example in the productionof diabodies) or detectable/functional labels as discussed in moredetail elsewhere herein.

Although in some aspects of the invention, binding members comprise apair of VH and VL domains, single binding domains based on either VH orVL domain sequences form further aspects of the invention. It is knownthat single immunoglobulin domains, especially VH domains, are capableof binding target antigens in a specific manner. For example, see thediscussion of dAbs above.

In the case of either of the single binding domains, these domains maybe used to screen for complementary domains capable of forming atwo-domain binding member able to bind A-FN and/or the ED-A offibronectin. This may be achieved by phage display screening methodsusing the so-called hierarchical dual combinatorial approach asdisclosed in WO92/01047, herein incorporated by reference in itsentirety, in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain binding member isselected in accordance with phage display techniques such as thosedescribed in that reference. This technique is also disclosed in Marks1992.

Binding members of the present invention may further comprise antibodyconstant regions or parts thereof, e.g. human antibody constant regionsor parts thereof. For example, a VL domain may be attached at itsC-terminal end to antibody light chain constant domains including humanCK or C chains, e.g. C2. Similarly, a binding member based on a VHdomain may be attached at its C-terminal end to all or part (e.g. a CH1domain) of an immunoglobulin heavy chain derived from any antibodyisotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes,particularly IgG1 and IgG4. Any synthetic or other constant regionvariant that has these properties and stabilizes variable regions isalso useful in embodiments of the present invention.

Binding members of the invention may be labelled with a detectable orfunctional label. A label can be any molecule that produces or can beinduced to produce a signal, including but not limited to fluorescers,radiolabels, enzymes, chemiluminescers or photosensitizers. Thus,binding may be detected and/or measured by detecting fluorescence orluminescence, radioactivity, enzyme activity or light absorbance.Detectable labels may be attached to antibodies of the invention usingconventional chemistry known in the art.

There are numerous methods by which the label can produce a signaldetectable by external means, for example, by visual examination,electromagnetic radiation, heat, and chemical reagents. The label canalso be bound to another binding member that binds the antibody of theinvention, or to a support.

Labelled binding members, e.g. scFv labelled with a detectable label,may be used diagnostically in vivo, ex vivo or in vitro, and/ortherapeutically.

For example, radiolabelled binding members (e.g. binding membersconjugated to a radioisotope) may be used in radiodiagnosis andradiotherapy. Radioisotopes which may be conjugated to a binding memberof the invention include isotopes such as ^(94m)Tc, ^(99m)Tc, ¹⁶⁶Re,¹⁸⁸Re, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ¹¹¹In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y,¹²¹Sn, ¹⁶¹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁰⁵Rh and ¹⁷⁷Lu.

For example, a binding member of the invention labelled with adetectable label may be used to detect, diagnose or monitor tumourmetastases and/or tumour metastasis in a human or animal. The bindingmember may be administered to a human or animal, normally a humanpatient, and the presence or absence of the antibody at a site distantfrom a site currently or previously occupied by a primary tumour in thehuman or animal body may be determined; localisation of the antibodymolecule to a site distant from the site currently or previouslyoccupied by the primary tumour in the human or animal indicates thepresence of a tumour metastases and/or tumour metastasis.

A binding member of the present invention may be used for themanufacture of a diagnostic product for use in diagnosing tumourmetastases.

The present invention also provides a method of detecting or diagnosinga tumour metastases in a human or animal comprising the steps of:

(a) administering to the human or animal a binding member of the presentinvention, for example labelled with a detectable label, which binds theED-A isoform of fibronectin and/or the ED-A of fibronectin, and(b) determining the presence or absence of the binding member at a sitedistant from a site currently or previously occupied by a primary tumourin the human or animal body;wherein localisation of the binding member to a site distant from thesite currently or previously occupied by the primary tumour in the humanor animal indicates the presence of a tumour metastases. Where thebinding member is labelled with a detectable label, the presence orabsence of the detectable label may be determined by detecting thelabel.

A binding member as described herein may also be used for measuringantigen levels in a competition assay, that is to say a method ofmeasuring the level of antigen in a sample by employing a binding memberas provided by the present invention in a competition assay. This may bewhere the physical separation of bound from unbound antigen is notrequired. Linking a reporter molecule to the binding member so that aphysical or optical change occurs on binding is one possibility. Thereporter molecule may directly or indirectly generate detectablesignals, which may be quantifiable. The linkage of reporter moleculesmay be directly or indirectly, covalently, e.g. via a peptide bond ornon-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

Competition assays can also be used in epitope mapping. In one instanceepitope mapping may be used to identify the epitope bound by a bindingmember. Such an epitope can be linear or conformational. Aconformational epitope can comprise at least two different fragments ofA-FN or the ED-A of fibronectin, wherein said fragments are positionedin proximity to each other when A-FN or the ED-A of fibronectin isfolded in its tertiary or quaternary structure to form a conformationalepitope which is recognized by a A-FN or the ED-A of fibronectin bindingmember. In testing for competition a peptide fragment of the antigen maybe employed, especially a peptide including or consisting essentially ofan epitope of interest. A peptide having the epitope sequence plus oneor more amino acids at either end may be used. Binding members accordingto the present invention may be such that their binding for antigen isinhibited by a peptide with or including the sequence given.

Further aspects of the present invention employ a conjugate or fusionbetween a binding member of the invention and a molecule that exerts abiocidal or cytotoxic effect on target cells in the lesions and anantibody directed against an extracellular matrix component which ispresent in such lesions. For example, the biocidal or cytotoxic moleculemay be interleukin-2 (IL-2), doxorubicin, interleukin-12 (IL-12),Interferon-γ (IFN-γ), Tumour Necrosis Factor α (TNFα) or tissue factor(preferably truncated). Such conjugates may be used therapeutically,e.g. for treatment of tumour metastases and/or tumour as referred toherein. Production and use of fusions or conjugates of binding memberswith biocidal or cytotoxic molecules is described for example inWO01/62298, which is incorporated by reference herein.

In one aspect the invention provides a method of treating tumourmetastasis and/or tumour metastases, the method comprising administeringa to an individual a therapeutically effective amount of a medicamentcomprising a binding member of the invention. The binding member may bea conjugate of (i) a molecule which exerts a biocidal or cytotoxiceffect on target cells by cellular interaction and (ii) a binding memberfor the ED-A isoform of fibronectin and/or the ED-A of fibronectin.

In another aspect the invention provides the use of a binding member ofthe invention for the preparation of a medicament for the treatment oftumour metastases and/or tumour metastasis. The binding member may be aconjugated or fused to a molecule that exerts a biocidal or cytotoxiceffect as described herein. The binding member may be a conjugate of (i)a molecule which exerts a biocidal or cytotoxic effect on target cellsby cellular interaction and (ii) a binding member for human fibronectinaccording to the present invention.

In a further aspect the invention provides a conjugate of (i) a moleculewhich exerts a biocidal or cytotoxic effect on target cells by cellularinteraction and (ii) a binding member for human fibronectin according tothe present invention, for use in a method of treatment of the human oranimal body by therapy. Such treatment may be of tumour metastasesand/or tumour metastasis.

A still further aspect of the invention provides a conjugate of (i) amolecule which exerts a biocidal or cytotoxic effect on target cells bycellular interaction and (ii) a binding member for human fibronectinaccording to the present invention. Such a conjugate preferablycomprises a fusion protein comprising the biocidal or cytotoxic moleculeand a said binding member, or, where the binding member is two-chain ormulti-chain, a fusion protein comprising the biocidal or cytotoxicmolecule and a polypeptide chain component of said binding member.Preferably the binding member is a single-chain polypeptide, e.g. asingle-chain antibody molecule, such as scFv. Thus a further aspect ofthe present invention provides a fusion protein comprising the biocidalor cytotoxic molecule and a single-chain Fv antibody molecule of theinvention.

The biocidal or cytotoxic molecule that exerts its effect on targetcells by cellular interaction, may interact directly with the targetcells, may interact with a membrane-bound receptor on the target cell orperturb the electrochemical potential of the cell membrane. Moleculeswhich interact with a membrane-bound receptor include chemokines,cytokines and hormones. Compounds which perturb the electrochemicalpotential of the cell membrane include hemolysin, ionophores, drugsacting on ion channels. In exemplary preferred embodiments the moleculeis interleukin-2, tissue factor (preferably truncated) or doxorubicin.Other embodiments may employ interleukin 12, interferon-gamma, IP-10 andTumor Necrosis Factor-α (TNF-α).

As discussed further below, the specific binding member is preferably anantibody or comprises an antibody antigen-binding site. Conveniently,the specific binding member may be a single-chain polypeptide, such as asingle-chain antibody. This allows for convenient production of a fusionprotein comprising single-chain antibody and the biocidal or cytotoxicmolecule (e.g. interleukin-2 or tissue factor). In other embodiments, anantibody antigen-binding site is provided by means of association of anantibody VH domain and an antibody VL domain in separate polypeptides,e.g. in a complete antibody or in an antibody fragment such as Fab ordiabody. Where the specific binding member is a two-chain or multi-chainmolecule (e.g. Fab or whole antibody, respectively), the biocidal orcytotoxic molecule may be conjugated as a fusion polypeptide with one ormore polypeptide chains in the specific binding member.

The binding member may be conjugated with the biocidal or cytotoxicmolecule by means of a peptide bond, i.e. within a fusion polypeptidecomprising said molecule and the specific binding member or apolypeptide chain component thereof. See Taniguchi et al. (1983) Nature302, 305-310; MaED-A et al. (1983) Biochem. Biophys. Res. Comm. 115:1040-1047; Devos et al. (1983) Nucl. Acids Res. 11: 4307-4323 for IL-2sequence information useful in preparation of a fusion polypeptidecomprising IL-2. Sequence information for truncated tissue factor isprovided by Scarpati et al. (1987) Biochemistry 26: 5234-5238, and Rufet al. (1991) J. Biol. Chem. 226: 15719-15725. Other means forconjugation include chemical conjugation, especially cross-linking usinga bifunctional reagent (e.g. employing DOUBLE-REAGENTS™ Cross-linkingReagents Selection Guide, Pierce).

Where slow release is desirable, e.g., where the biocidal or cytotoxicmolecule is doxorubicin or other molecule which perturbs theelectrochemical potential of the cell membrane, chemical conjugation maybe by means of formation of a Schiff base (imine) between a primaryamino group of the specific binding member (a polypeptide such as anantibody or antibody fragment) and an oxidised sugar moiety(daunosamine) of the biocidal or cytotoxic molecule such as doxorubicin.

The present invention further provides an isolated nucleic acid encodinga binding member of the present invention. Nucleic acid may include DNAand/or RNA. In one aspect, the present invention provides a nucleic acidthat codes for a CDR or set of CDRs or VH domain or VL domain orantibody antigen-binding site or antibody molecule, e.g. scFv or IgG,e.g. IgG1, of the invention as defined above. Preferred nucleotidesequences are the nucleotide sequences encoding VH and/or VL domainsdisclosed herein.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell thatcomprises one or more constructs as above. A nucleic acid encoding anyCDR or set of CDRs or VH domain or VL domain or antibody antigen-bindingsite or antibody molecule, e.g. scFv or IgG1 or IgG4 as provided, itselfforms an aspect of the present invention, as does a method of productionof the encoded product, which method comprises expression from encodingnucleic acid. Expression may conveniently be achieved by culturing underappropriate conditions recombinant host cells containing the nucleicacid. Following production by expression a VH or VL domain, or bindingmember may be isolated and/or purified using any suitable technique,then used as appropriate.

Nucleic acid according to the present invention may comprise DNA or RNAand may be wholly or partially synthetic. Reference to a nucleotidesequence as set out herein encompasses a DNA molecule with the specifiedsequence, and encompasses a RNA molecule with the specified sequence inwhich U is substituted for T, unless context requires otherwise.

A yet further aspect provides a method of production of an antibody VHvariable domain, the method including causing expression from encodingnucleic acid. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

Analogous methods for production of VL variable domains and bindingmembers comprising a VH and/or VL domain are provided as further aspectsof the present invention.

A method of production may comprise a step of isolation and/orpurification of the product. A method of production may compriseformulating the product into a composition including at least oneadditional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, filamentous fungi, yeast andbaculovirus systems and transgenic plants and animals. The expression ofantibodies and antibody fragments in prokaryotic cells is wellestablished in the art. For a review, see for example Plückthun 1991. Acommon bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of a binding member forexample Chadd & Chamow (2001), Andersen & Krummen (2002), Larrick &Thomas (2001). Mammalian cell lines available in the art for expressionof a heterologous polypeptide include Chinese hamster ovary (CHO) cells,HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0rat myeloma cells, human embryonic kidney cells, human embryonic retinacells and many others.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids e.g.phagemid, or viral e.g. ‘phage, as appropriate. For further details see,for example, Sambrook & Russell (2001). Many known techniques andprotocols for manipulation of nucleic acid, for example in preparationof nucleic acid constructs, mutagenesis, sequencing, introduction of DNAinto cells and gene expression, and analysis of proteins, are describedin detail in Ausubel 1999.

A further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. Such a host cell may be invitro and may be in culture. Such a host cell may be in vivo. In vivopresence of the host cell may allow intracellular expression of thebinding members of the present invention as “intrabodies” orintracellular antibodies. Intrabodies may be used for gene therapy.

A still further aspect provides a method comprising introducing nucleicacid of the invention into a host cell. The introduction may employ anyavailable technique. For eukaryotic cells, suitable techniques mayinclude calcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using retrovirus orother virus, e.g. vaccinia or, for insect cells, baculovirus.Introducing nucleic acid in the host cell, in particular a eukaryoticcell may use a viral or a plasmid based system. The plasmid system maybe maintained episomally or may incorporated into the host cell or intoan artificial chromosome. Incorporation may be either by random ortargeted integration of one or more copies at single or multiple loci.For bacterial cells, suitable techniques may include calcium chloridetransformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene. The purification of the expressed product may beachieved by methods known to one of skill in the art.

In accordance with the invention, the nucleic acid of the invention maybe integrated into the genome (e.g. chromosome) of the host cell.Integration may be promoted by inclusion of sequences that promoterecombination with the genome, in accordance with standard techniques.

The present invention also provides a method that comprises using aconstruct as stated above in an expression system in order to express abinding member or polypeptide as above.

Binding members of the present invention are designed to be used inmethods of diagnosis or treatment in human or animal subjects, e.g.human. Binding members may be used in diagnosis or treatment of tumourmetastases and/or tumour metastasis.

Accordingly, further aspects of the invention provide methods oftreatment comprising administration of a binding member as provided,pharmaceutical compositions comprising such a binding member, and use ofsuch a binding member in the manufacture of a medicament foradministration, for example in a method of making a medicament orpharmaceutical composition comprising formulating the binding memberwith a pharmaceutically acceptable excipient. Pharmaceuticallyacceptable vehicles are well known and will be adapted by the personskilled in the art as a function of the nature and of the mode ofadministration of the active compound(s) chosen.

Binding members of the present invention will usually be administered inthe form of a pharmaceutical composition, which may comprise at leastone component in addition to the binding member. Thus pharmaceuticalcompositions according to the present invention, and for use inaccordance with the present invention, may comprise, in addition toactive ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, inhaled or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration such as for examplenanobodies etc are also envisaged in the present invention. Such oralformulations may be in tablet, capsule, powder, liquid or semi-solidform. A tablet may comprise a solid carrier such as gelatin or anadjuvant. Liquid pharmaceutical compositions generally comprise a liquidcarrier such as water, petroleum, animal or vegetable oils, mineral oilor synthetic oil. Physiological saline solution, dextrose or othersaccharide solution or glycols such as ethylene glycol, propylene glycolor polyethylene glycol may be included.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be employed, as required. Many methods for thepreparation of pharmaceutical formulations are known to those skilled inthe art. See, e.g., Robinson, 1978.

A composition may be administered alone or in combination with othertreatments, concurrently or sequentially or as a combined preparationwith another therapeutic agent or agents, dependent upon the conditionto be treated.

A binding member for A-FN and/or the ED-A of fibronectin may be used aspart of a combination therapy in conjunction with an additionalmedicinal component. Combination treatments may be used to providesignificant synergistic effects, particularly the combination of abinding member which binds the A-FN and/or the ED-A of fibronectin withone or more other drugs. A binding member for the A-FN and/or the ED-Aof fibronectin may be administered concurrently or sequentially or as acombined preparation with another therapeutic agent or agents, for thetreatment of one or more of the conditions listed herein.

For example, a binding member of the invention may be used incombination with an existing therapeutic agent for the treatment oftumour metastases and/or tumour metastasis. Existing therapeutic agentsfor the treatment of tumour metastases and/or tumour metastasis include:doxorubicin, taxol, gemcitabine, sorafenib, melphalan, and avastin.

A binding member of the invention and one or more of the aboveadditional medicinal components may be used in the manufacture of amedicament. The medicament may be for separate or combinedadministration to an individual, and accordingly may comprise thebinding member and the additional component as a combined preparation oras separate preparations. Separate preparations may be used tofacilitate separate and sequential or simultaneous administration, andallow administration of the components by different routes e.g. oral andparenteral administration.

In accordance with the present invention, compositions provided may beadministered to mammals. Administration may be in a “therapeuticallyeffective amount”, this being sufficient to show benefit to a patient.Such benefit may be at least amelioration of at least one symptom. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe composition, the type of binding member, the method ofadministration, the scheduling of administration and other factors knownto medical practitioners. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and may depend on the severity of the symptomsand/or progression of a disease being treated.

Appropriate doses of antibody are well known in the art (Ledermann 1991and Bagshawe 1991. Specific dosages indicated herein, or in thePhysician's Desk Reference (2003) as appropriate for the type ofmedicament being administered, may be used. A therapeutically effectiveamount or suitable dose of a binding member of the invention can bedetermined by comparing its in vitro activity and in vivo activity in ananimal model. Methods for extrapolation of effective dosages in mice andother test animals to humans are known. The precise dose will dependupon a number of factors, including whether the antibody is fordiagnosis, prevention or for treatment, the size and location of thearea to be treated, the precise nature of the antibody (e.g. wholeantibody, fragment or diabody), and the nature of any detectable labelor other molecule attached to the antibody. A typical antibody dose willbe in the range 100 μg to 1 g for systemic applications, and 1 μg to 1mg for topical applications. An initial higher loading dose, followed byone or more lower doses, may be administered. An antibody may be a wholeantibody, e.g. the IgG1 or IgG4 isotype. This is a dose for a singletreatment of an adult patient, which may be proportionally adjusted forchildren and infants, and also adjusted for other antibody formats inproportion to molecular weight. Treatments may be repeated at daily,twice-weekly, weekly or monthly intervals, at the discretion of thephysician. Treatments may be every two to four weeks for subcutaneousadministration and every four to eight weeks for intravenousadministration. In some embodiments of the present invention, treatmentis periodic, and the period between administrations is about two weeksor more, e.g. about three weeks or more, about four weeks or more, orabout once a month. In other embodiments of the invention, treatment maybe given before, and/or after surgery, and may be administered orapplied directly at the anatomical site of surgical treatment.

Further aspects and embodiments of the invention will be apparent tothose skilled in the art given the present disclosure including thefollowing experimental exemplification.

EXPERIMENTAL Materials and Methods Animal Model

Animal experiments were approved by the Swiss Federal Veterinary Officeand performed in accordance with the Swiss Animal Protection Ordinance.Mice were monitored regularly. When showing any sign of pain orsuffering, or in case of a body weight loss >15% animals wereeuthanized. Male Sv129 mice (RCC, Füllingsdorf, Switzerland) receivedintravenous injection of ˜106 mutant F9 murine teratocarcinoma cells(Terrana et al. 1987), which had been kindly provided by Dario Rusciano(SIFI, Catania, Italy). Mice were used 3 weeks after tumour cellinjection for in vivo biotinylation, targeting experiments or organexcision for immunohistochemistry.

In Vivo Biotinylation

In vivo biotinylation experiments were performed as described previously(Roesli et al. 2006, Rybak et al. 2005). In brief, the chest of theanesthetized mouse was opened through a median sternotomy. The leftheart ventricle was punctured with a perfusion needle and a small cutwas made in the right atrium to allow the outflow of the perfusionsolutions. Immediately after, perfusion of the systemic circulation wasperformed with a pressure of 100 mm Hg at a flow rate of 1.5 ml/min. Ina first step, perfusion was carried out with 15 ml biotinylationsolution (prewarmed to 38° C.), containing 1 mg/ml sulfo-NHS-LC-biotin(Pierce, Rockford, Ill., USA) in PBS, pH 7.4, supplemented with 10%(w/v) dextran-40 (Amersham Biosciences, Uppsala, Sweden) as plasmaexpander. Thereby, blood components, which could compete with thebiotinylation reaction, were eliminated from circulation within thefirst few minutes of perfusion and accessible primary amine-containingproteins (and certain glycolipids and phospholipids) in the differenttissues could be covalently modified with biotin. To neutralizeunreacted biotinylation reagent, the in vivo biotinylation was followedby a 10 min washing step with 50 mM Tris, 10% (w/v) dextran-40, in PBS,pH 7.4, prewarmed to 38° C. During perfusion with biotinylation reagent(and during the first three minutes of the following perfusion withquenching solution) the region around the heart was washed with 50 mMTris in PBS, pH 7.4 (38° C.), to quench out-flowing unreactedbiotinylation reagent and avoid undesired labelling of molecules at theorgan surfaces. After perfusion, organs and tumours were excised andspecimens were either freshly snap-frozen for preparation of organhomogenates or embedded in cryoembedding compound (Microm, Walldorf,Germany) and frozen in isopentane in liquid nitrogen for preparation ofcryosections for histochemical analysis. Unperfused mice were used asnegative controls for the proteomic analysis.

Preparation of Protein Extracts for Proteomic Analysis

Specimens were resuspended in 40 μl per mg tissue of lysis buffer (2%SDS, 50 mM Tris, 10 mM EDTA, Complete E proteinase inhibitor cocktail(Roche Diagnostics, Mannheim, Germany) in PBS, pH 7.4) and homogenizedusing an Ultra-Turrax T8 disperser (IKA-Werke, Staufen, Germany).Homogenates were sonicated using a Vibra-cell (Sonics, New Town, Conn.,USA), followed by 15 min incubation at 99° C. and 20 min centrifugationat 15000×g. The supernatant was used as total protein extract. Proteinconcentration was determined using the BCA Protein Assay Reagent Kit(Pierce).

Purification of Biotinylated Proteins

For each sample, 960 μl streptavidin-sepharose (Amersham Biosciences,Uppsala, Sweden) slurry were washed three times in buffer A (NP40 1%,SDS 0.1% in PBS), pelleted and mixed with 15 milligrams of total proteinextract. Capture of biotinylated proteins was allowed to proceed for 2 hat RT in a revolving mixer. The supernatant was removed and the resinwashed three times with buffer A, two times with buffer B (NP40 0.1%,NaCl 1 M in PBS), and once with 50 mM ammonium bicarbonate. Finally, theresin was resuspended in 400 μl of a 50 mM solution of ammoniumbicarbonate and 20 μl of sequencing grade modified porcine trypsin(stock solution of 40 ng/μl in 50 mM ammonium bicarbonate) (Promega,Madison, Wis., USA) were added. Protease digestion was carried outovernight at 37° C. under constant agitation. Peptides were desalted,purified and concentrated with C18 microcolumns (ZipTip C18, Millipore,Billerica, Mass., USA). After lyophilisation peptides were stored at−20° C.

Nano Capillary-HPLC with Automated Online Fraction Spotting onto MALDITarget Plates

Tryptic peptides were separated by reverse phase high Performance liquidchromatography (RP-HPLC) using an UltiMate nanoscale LC system and aFAMOS microautosampler (LC Packings, Amsterdam, The Netherlands)controlled by the Chromeleon software (Dionex, Sunnyvale, Calif., USA).Mobile phase A consisted of 2% acetonitrile and 0.1% trifluoroaceticacid (TFA) in water, mobile phase B was 80% acetonitrile and 0.1% TFA inwater. The flow rate was 300 nl/min. Lyophilized peptides derived fromthe digestion of biotinylated proteins affinity purified from 1.5 mg oftotal protein were dissolved in 5 μl of buffer A and loaded on thecolumn (inner diameter: 75 μm, length 15 cm, filled with C18 PepMap 100,3 μm, 100 Å beads; LC Packings). The peptides were eluted with agradient of 0-30% B for 7 min, 30-80% B for 67 min, 80-100% B for 3 minand 100% B for 5 min; the column was equilibrated with 100% A for 20 minbefore analyzing the next sample. Eluting fractions were mixed with asolution of 3 mg/ml cyano-4-hydroxy cinnamic acid, 277 μmol/mlneurotensin (internal standard), 0.1% TFA, and 70% acetonitrile in waterand deposed on a 192-well MALDI target plate using an on-line Probotsystem (Dionex). The flow of the MALDI-matrix solution was set to 1.083μl/min. Thus, each fraction collected during 20 s contained 361 nlMALDI-matrix solution and 100 nl sample. The end-concentration ofneurotensin was 100 fmol per well.

MALDI-TOF/TOF Mass Spectrometry

MALDI-TOF/TOF mass spectrometric analysis was carried out with the 4700Proteomics Analyzer (Applied Biosystems, Framingham, Mass., USA). Forprecursor ion selection, all fractions were measured in MS mode beforeMS/MS was performed. A maximum of 15 precursors per sample spot wereselected for subsequent fragmentation by collision induced dissociation.Spectra were processed and analyzed by the Global Protein ServerWorkstation (Applied Biosystems), which uses internal MASCOT (MatrixScience, London, UK) software for matching MS and MS/MS data againstdatabases of in silico digested proteins. The data obtained werescreened against a mouse database downloaded from the NCBI homepage(http://www.ncbi.nlm.nih.gov/). Protein identifications, performed bymeans of the MASCOT software, were considered to be correct calls withinthe 95% confidence interval for the best peptide ion.

MALDI-TOF and MALDI-TOF/TOF mass spectrometric analyses were carried outusing the 4700 Proteomics Analyzer (Applied Biosystems). Peptide masseswere acquired over a range from 750 to 4000 m/z, with a focus mass of2000 m/z. MS spectra were summed from 2000 laser shots from an Nd:YAGlaser operating at 355 nm and 200 Hz. An automated plate calibration wasperformed using five peptide standards (masses 900-2400 m/z; AppliedBiosystems) in six calibration wells. This plate calibration was used toupdate the instrument default mass calibration, which was applied to allMS and MS/MS spectra. Furthermore, an internal calibration of each MSspectrum using the internal standard peptide added to the MALDI matrixwas performed. A maximum of 15 precursors per sample well with asignal-to-noise ratio of >100 was automatically selected for subsequentfragmentation by collision induced dissociation. MS/MS spectra weresummed from 2500 to 5000 laser shots. Spectra were processed andanalyzed by the Global Protein Server Workstation (Applied Biosystems),which uses internal MASCOT (Matrix Science) software for matching MS andMS/MS data against databases of in-silico digested proteins. The MASCOTsearch parameters were (i) a mouse database downloaded from the EuropeanBioinformatics Institute (EBI) homepage on the 9 Sep. 2006(ftp.ebi.ac.uk/pub/databases/SPproteomes/fasta/proteomes/59.M_musculus.fasta.gz);(ii) enzyme: trypsin and semi-trypsin; (iii) allowed number of missedcleavages: 1; (iv) variable posttranslational modifications: methionineoxidation; (v) peptide tolerance: ±30 ppm; (vi) MS/MS tolerance: ±0.2Da; (vii) peptide charge: +1; (viii) minimum ion score C.I. % forpeptides: 95 and (ix) maximum peptide rank: 1. Furthermore, an MS/MSpeak filtering with the following parameters was used: (i) mass range:60 Da to 20 Da below precursor mass; (ii) minimum signal-to-noise ratio:6; (iii) peak density filter: maximum 30 peaks per 200 Da and (iv)maximum number of peaks per spectrum: 65.

Antibodies

The isolation of the anti-ED-B antibody fragment scFv(L19) has beenpreviously described (Pini et al. 1998). The parent anti-ED-A antibodywas isolated from the ETH-2 library using published procedures(Giovannoni, Nucleic. Acid Research, 2001, 29(5):E27). The affinitymaturation of the parent anti-ED-A antibody, yielding the high affinityanti-ED-A antibodies, is described in the following section.

Affinity Maturation of the Parent Anti-ED-A Antibody

The parent anti-ED-A antibody (an ETH-2-derived antibody) was used astemplate for the construction of an affinity maturation library.Sequence variability in the VH CDR1 (DP47 germline) and VL CDR1 (DPK22germline) of the library was introduced by PCP using partiallydegenerate primers 5′-CTGGAGCCTGGCGGACCCAGCTCATMNNMNNMNNGCTAAAGGTGAATCCAGA-3′ (SEQ ID NO: 17) for VH and 5′-CCAGGTTTCTGCTGGTACCAGGCTAAMNNMNNMNNGCTAACACTCTGACTGGCCCTGC-3′ (SEQ ID NO: 18) for VL (alloligonucleotides were purchased from Operon Biotechnologies, Cologne,Germany), in a process that generates random mutations at positions 31,32 and 33 of the VH CDR1 and at positions 31, 31a and 32 of the VL CDR1.VHVL combinations were assembled in scFv format by PCR assembly usingthe primers LMB3long (5′-CAGGAAACAGCTATGACCATGATTAC-3′) (SEQ ID NO: 19)and fdseqlong (5′-GACGTTAGTAAATGAATTTTCTGTATGAGG-3′) (SEQ ID NO: 20),using gel-purified VH and VL segments as templates. The assembled VH-VLfragments were doubly digested with NcoI/NotI and cloned intoNcoI/NotI-digested pHEN1 phagemid vector (Hoogenboom et al., 1991). Theresulting ligation product was electroporated into electrocompetent E.coli TG-1 cells according to (Viti et al., 2000), giving rise to alibrary containing 1.5×10⁷ individual antibody clones, which wasscreened for antibodies which bind ED-A with improved affinity.

Selection of Anti-ED-A Antibodies

The antibody library described above was screened for antibodies whichbound ED-A with a greater affinity than the parent anti-ED-A antibodyusing BIAcore analysis. The antigen (11A12) used in the BIAcore analysiscontained the ED-A domain of human fibronectin and has the followingamino acid sequence (SEQ ID NO: 120):

MRSYRTEIDKPSQMQVTDVQDNSISVKWLPSSSPVTGYRVTTTPKNGPGPTKTETAGPDQTEMTIEGLQPTVEYVVSVYAQNPSGESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVRSHHHHHH

The nucleotide sequence of antigen (11A12) (SEQ ID NO: 121) is asfollows:

atgagatcctaccgaacagaaattgacaaaccatcccagatgcaagtgaccgatgttcaggacaacagcattagtgtcaagtggctgccttcaagttcccctgttactggttacagagtaaccaccactcccaaaaatggaccaggaccaacaaaaactaaaactgcaggtccagatcaaacagaaatgactattgaaggcttgcagcccacagtggagtatgtggttagtgtctatgctcagaatccaagcggagagagtcagcctcttccatcaaaattgcttgggaaagcccacaggggcaagtttccaggtacagggtgacctactcgagccctgaggatggaatccatgagctattccctgcacctgatggtgaagaagacactgcagagctgcaaggcctcagaccgggttctgagtacacagtcagtgtggttgccttgcacgatgatatggagagccagcccctgattggaacccagtccacagctattcctgcaccaactgacctgaagttcactcaggtcacacccacaagcctgagcgcccagtggacaccacccaatgttcagctcactggatatcgagtgcgggtgacccccaaggagaagaccggaccaatgaaagaaatcaaccttgctcctgacagctcatccgtggttgtatcaggacttatggtggccaccaaatatgaagtgagtgtctatgctcttaaggacactttgacaagcagaccagctcagggagttgtcaccactctggagaatgtcagatctcatcaccatcaccatcactaa

The nucleotide sequence of the antigen was amplified by FOR usingprimers containing BamHI and BglII restriction sites at the 5′ and 3′respectively. The resulting PCR product and the vector pQE12 (QIAGEN)were digested with BamHI and BglII restriction endonuclease andsubsequently ligated in a reaction containing a ratio of insert tovector of 3:1. The resulting vector was sequenced to check that thesequence was correct.

The antigen was prepared as follows:

A TG1 electrocompetent Preculture in 10 ml 2TY, Amp, 1% Glucose waselectroporated in the presence of 1 μl of a DNA miniprep of 11A12. Thepre-culture was then diluted 1:100 (8 ml in 800 ml of 2TY, Amp, 0.1%Glucose) and grown to an OD600 of 0.4-0.6 and then induced with IPTGover night. The following day the cells were spun down and thesupernatant filtered (Millipore 0.22 μm). After centrifugation andclarification of the culture broth, 11A12 was purified using a Hitrapcolumn on FPLC. The Ni/column was regenerated as follows: the column wasrinsed with 5 column volumes (CV) H2O followed by application of 3 CV0.5 M EDTA/0.2 M Tris pH 8 to wash the old Nickel out from the column.This was followed by rinsing of the column with 5 CV H2O. The column wasthen reloaded with 2 CV 100 mM NiSO4 followed by rinsing of the columnwith several CVs H2O. The column was then equilibrated with 5 CV lysisbuffer (20 mM imidazol/250 mM NaCl/PBS pH 7.4). The cell lysate wasfiltered (Millipore 0.45 μm) and loaded onto the column (manually). Thecolumn was then put back on FPLC and the lysis buffer left to flow untilthe UV signal was stable (constant), about 3 CV. The elution program wasthen started: Gradient from 0% to 100% of Elution Buffer (400 mMimidazol/250 mM NaCl/PBS pH 7.4) in 5 CV. The fractions containing theeluted antigen were pooled and dialysed in PBS over night.

Expression and Purification of the Anti-ED-A Antibodies

The anti-ED-A antibodies were expressed and purified as follows: A TG1electrocompetent Preculture in 10 ml 2TY, Amp, 1% Glucose waselectroporated in the presence of 1 μl of a DNA miniprep of one of theanti-ED-A antibodies. The pre-culture was then diluted 1:100 (8 ml in800 ml of 2TY, Amp, 0.1% Glucose) and grown to an OD600 of 0.4-0.6 andthen induced with IPTG over night. The following day the cells were spundown and the supernatant filtered (Millipore 0.22 μm). The scFv werepurified on a Protein A-Sepharose column and Triethylemmine was used toelute the scFvs from the column. The fractions containing the elutedscFvs were dialysed in PBS over night at 4° C. The scFv fractions werethen put on a Superdex 75 column with PBS flowing at 0.5 ml/min and 0.25ml fractions collected. The monomeric fractions were used for BIAcoreanalysis.

BIAcore Analysis 1

The BIAcore Chip was flushed overnight at a flow rate of 5 μl/min withHBS-EP buffer BIACORE, 0.01 M Hepes pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% surfactant P20 (same buffer used for the assay). The antigen(11A12) was diluted to a concentration of 50 μg/ml in acetate buffer (pH4.0) and the COOH groups on the chip were activated by injection of 50μl of a mix of N-Hydroxy Succinimmide (NHS) andethyl-N-(dimethylaminopropyl)-carbodiimide (EDC). 40 μl of the 11A12antigen were injected onto the chip and the residual free COOH groupswere blocked with 30 μl of ethanolamine. After a 0.22 μm filtration, 20μl of each individual bacterial supernatant were injected onto the chipand interaction with the antigen was monitored in real time.

BIAcore Analysis 2

The k_(on), k_(off) and K_(D) of the parent anti-ED-A antibody andanti-ED-A antibodies B2, C5, D5, C8, F8, B7 and G9 were evaluated usingSurface Plasmon Resonance. The chip was equilibrated over night with thesame buffer used during the assay at a buffer flow rate of 5 μl/min. Thewhole coating procedure was performed at this flow rate. The antigen11A12 was diluted 1:25 with acetate buffer pH 4.00 (provided by BIACORE)to a final concentration of 20 μg/ml. The NHS and EDC were then mixedand 50 μl injected to activate the COOH groups on the CM5 chip. This wasfollowed by injection of 40 μl of the antigen (this lasts about 40″).Then 30 μl of Ethanolammine were injected in order to block thereactivity of eventual free COOH.

Each sample was assayed at a flow rate 20 μl/min. 20 μl of undilutedmonomeric protein (as it comes out from the gel filtration) wasinjected. The dissociation time was left to run for about 200″. Then 10μl of HCl 10 mM was injected to regenerate the chip. The injection ofmonomeric protein was repeated at different dilutions, i.e. 1:2 dilution(in PBS) followed by regeneration with HCl. This was followed by a thirdinjection of the protein, at a dilution of 1:4 followed again byregeneration with HCl. The k_(on), k_(off) and KD values for eachanti-ED-A antibody were evaluated using the BIAevaluation software.

Histochemistry

In order to verify successful in vivo biotinylation, staining ofbiotinylated structures after was performed as described in (Rybak etal. 2005). Section (10 μm) were cut from freshly-frozen specimens, fixedwith acetone, incubated successively with streptavidin:biotinylatedalkaline phosphatase complex (Biospa, Milano, Italy) and with Fast-RedTR (Sigma) [in the presence of 1 mM Levamisole to inhibit endogenousalkaline phosphatase] and counterstained with Hematoxylin solution(Sigma). Immunohistochemical staining with scFv-antibodies, whichcarried a FLAG-tag, was performed as described earlier (see, e.g.,(Brack et al. 2006)). In brief, sections were incubated with the scFvfragments (final concentration, 2-10 μg/mL) and with monoclonalanti-Flag antibody M2 simultaneously. Bound antibodies were detectedwith rabbit anti-mouse immunoglobulin antibody (Dakocytomation,Glostrup, Denmark) followed by mouse monoclonal alkalinephosphatase-anti-alkaline phosphatase complex (Dakocytomation). Fast Red(Sigma) was used as phosphatase substrate, and sections werecounterstained with hematoxylin (Sigma).

All sections were mounted with Glycergel (DakoCytomation, Glostrup,Denmark) and analyzed with an Axiovert S100 TV microscope (Carl Zeiss,Feldbach, Switzerland) using the Axiovision software (Carl Zeiss).

In Vivo Targeting with Anti-ED-A Antibody

The parent anti-ED-A antibody scFv was labelled with the commerciallyavailable infrared fluorophore derivative Alexa Fluor 750 carboxylicacid succinididyl ester (Invitrogen) according to the provider'sprotocol. The labelled antibody was separated from the unreacted dye bygel filtration using a PD-10 column (GE Healthcare). The degree oflabelling, estimated according to the Invitrogen labelling protocol, was5 dye molecules per antibody molecule. The Alexa 750-labeled parentanti-ED-A scFv antibody (in a final concentration of 0.3 mg/ml) wasinjected (200 μl/mouse, i.e. 60 μg antibody/mouse) in the tail vein ofSv190 mice 3 weeks after injection of F9DR tumour cells. Mice organswere excised 6 hours after injection of the labelled antibody and imagedwith a home-built infrared fluorescence imager (Birchler et al. 1999)equipped with a tungsten halogen lamp, excitation and emission filtersspecific for Alexa 750, and a monochrome CCD camera.

Biodistribution of F8 Diabody

The F8 diabody comprises the same VH and VL domains as anti-ED-Aantibody F8, e.g. as employed in scFv format. The F8 diabody and theanti-ED-A scFv F8 have different linker sequences between the VH and theVL domains. The amino acid sequence of the F8 diabody linker is GSSGG(SEQ ID NO: 28) (nucleotide sequence: gggtccagtggcggt [SEQ ID NO: 29]).Therefore, the F8 diabody linker sequence is five amino acids long,while in the anti-ED-A scFv F8 the linker is 20 amino acids long (seeSEQ ID NO: 11). The reduction in the length of the linker between the VLand VH domains, means that intermolecular rather intramolecular pairingof VL and VH domains is favoured. Consequently, the VL domain of one F8polypeptide is more likely to pair with the VH domain of another F8polypeptide than it is to pair with the VH domain of the same F8polypeptide.

The F8 diabody was expressed in E. coli TG1 cells as follows: DNAencoding the F8 diabody was introduced into electrocompetent E. coli TG1cells using electroporation. The electroporated E. coli cells wereprecultured in 10 ml 2YT medium, Amp, 1% Glucose. The preculture wasdiluted 1:100 into 800 ml 2YT medium, Amp, 0.1% Glucose and the culturegrown to a density (OD600 nm) of 0.6. Expression of the F8 diabody wasthen induced using 1 mM of IPTG.

The expressed F8 diabody was labelled with ¹²⁵I as follows: 10 μl ofsterile PBS was added into an iodogen tube (coated with 50 μl 0.1 mg/mliodogen in Chloroform) followed by addition of 2 μl ¹²⁵I sodium iodide(˜200 μCi) and incubation at room temperature (RT) for 5 min.

400 μl of F8 diabody at an OD 0.2 (˜60 μg) were then added to theiodogen tube and incubated at RT for 25 min. 1/100 of this mixture wascollected in order to measure the radioactivity contained in the mixture(referred to as ‘INPUT’). The labelled F8 diabody was then loaded onto asize exclusion chromatography column (PD10: Sephadex G-25 M, GEHealthcare) in order to separate the iodinated F8 diabody from the freeiodine. The radioactivity of the collected iodinated F8 diabody wasmeasured and the percentage of iodine incorporated into the F8 diabodycalculated (CPM [counts per minute] of iodinated F8 diabody/CPM INPUT)to be between 30-40%.

Four F9 tumour bearing mice were put on Lugol for 2 days (600 μl into300 ml) in order to block the thyroid and each mouse injectedintravenously with 200 μl of the iodinate F8 diabody (about 5-8 μgiodinate F8 diabody [18 μCi] per mouse). After 24 hours the mice weresacrificed and tumour, liver, lung, spleen, heart, kidney, intestine,tail and blood were removed (referred to collectively herein in thiscontext as mouse ‘tissues’) and used for radioactive counting. The levelof radioactivity in each tissue sample was measured using a Perkingamma-counter. The ‘output’ was calculated by dividing the percentage ofthe injected dose (in CPM) by the weight of the tissue (in grams) (%ID/g).

Results Identification of Differentially Expressed Proteins and SpliceVariants

The perfusion-based chemical proteomic methodology used for thecomparative analysis of accessible proteins in liver and in F9 livermetastases (Terrana et al. 1987) is depicted in FIG. 1A. These tumoursdevelop large metastatic foci on the surface and inside the mouse liver(FIG. 1B). Under terminal anaesthesia, tumour-bearing mice were perfusedwith 15 ml of a 1.8 mM solution ofsulfosuccinimidyl-6-[biotin-amido]hexanoate (1 mg/ml) in PBS, pH 7.4,supplemented with 10% Dextran-40 as plasma expander. The procedure,which typically lasted 10 minutes, allowed the removal of blood from allthe organs of the major circulation and the selective biotinylation ofaccessible proteins, both on the luminal and abluminal aspects of bloodvessels. Virtually all blood vessels of F9 liver metastases wereefficiently and selectively labelled with this procedure, as confirmedby histochemical staining with a streptavidin-alkaline phosphataseconjugate (FIG. 1C). In the normal liver, blood vessels were stronglystained, but labelling of some sinusoids was also detected, compatiblywith the physiological filter function of the liver (FIG. 1C). The invivo biotinylation was quenched by perfusion with a solution containingprimary amines. Subsequently, specimens of liver metastases were excisedfrom the liver, homogenized and used for the recovery of biotinylatedproteins in the presence of the strong detergent SDS by affinitychromatography on streptavidin resin (FIG. 1A). In order to minimize therisk of diffusion of metastatic proteins in the host liver, liver fromin vivo biotinylated healthy mice was used for the study of the normalliver vasculature. The use of host liver from F9 tumour mice would havealso been problematic because of the little residual healthy tissue andbecause it would have been difficult to exclude macroscopically theabsence of micrometastases. In total, samples from 7 in vivobiotinylated healthy mice and 9 in vivo biotinylated F9 tumour-bearingmice were used for the proteomic analysis. In addition, specimens from 2healthy and 3 metastases-bearing non-biotinylated mice were used asnegative controls. Stringent washing procedures and on-resin trypticdigestion of streptavidin captured proteins from F9 metastases andnormal liver (processed in parallel) yielded a collection of peptides,which could be separated, identified and compared using nano-HPLC andMALDI-TOF/TOF mass spectrometric procedures (Roesli et al., 2006).

In total, 1291 different peptides were identified (>95% Mascotconfidence level) which were grouped by the Mascot software to 480different peptide sets. A few of these peptide sets were also found innegative control samples from non-biotinylated mice (like carboxylaseswhich carry endogenous biotin as a co-factor, keratins as contaminants,or very abundant proteins like serum albumin). Of the residual 435identified peptide sets, 331 could be annotated by the Mascot softwareunambiguously to a single protein, while 104 peptide sets were annotatedto multiple (in total to 358) proteins. In most cases, multiple proteinsannotated to the same peptide set belong to a related protein family(e.g., immunoglobulins) or can even be the same proteins with differentdatabase entries. Of the 435 different peptide sets, 117 wereexclusively found in metastasis specimens, 193 only in healthy liverspecimens and 125 in both types of tissues. For example, peptidesmatching to fibronectin (National Center for Biotechnology Information[NCBI] accession number P11276) were found in four healthy liverspecimens and eight metastasis specimens.

Proteins found in both the healthy liver and metastasis specimens (e.g.fibronectin) may be present at substantially different levels in the twosamples. If this was the case, this should be reflected in the number ofspecimens in which the proteins were detected, and/or in the number ofpeptides (as well as normalized peptide signal intensity; (Scheurer etal. 2005, Scheurer et al. 2005)) observed in the liver and metastasissamples. For instance, 38 tryptic peptides from fibronectin (NCBIaccession number P11276) were found only in metastasis specimens, whileone peptide was found only in healthy liver specimens. Eleven peptideswere found in both types of specimens.

The striking abundance of fibronectin-derived peptides detected in livermetastases, in spite of the fact that liver is the site of fibronectinbiosynthesis, prompted us to investigate differences in relativeabundance of fibronectin-derived peptides and the over-expression ofalternatively spliced domains. Table 1 lists all fibronectin peptidesidentified in the proteomic analysis. Mouse fibronectin, contains twotype-III globular extra-domains which may undergo alternative splicing:ED-A and ED-B (ffrench-Constant 1995, Hynes 1990, Kaspar et al. 2006).In addition, the IIICS segment undergoes different splicing patterns inmice and humans. Interestingly, all three ED-A-derived peptides as wellas the IIICS-derived peptide were observed only in the tumour samples.

ED-B-derived peptides would not be visible in this analysis due to thefact that ED-B contains no lysine residue and the two arginines giverise to peptides which are too large in size for detection. FIG. 2Ashows the location of the peptides identified in tumour specimens(Tumor) and healthy liver specimens (Normal) on the fibronectin domainstructure. FIG. 2B shows the relative intensity of normalized MS signalsfor two fibronectin-derived peptides: IAWESPQGQVSR (SEQ ID NO: 16) whichis located within the ED-A domain and FLTTTPNSLLVSWQAPR (SEQ ID NO: 15),which is located in domain 14. The latter peptide was more abundant inthe metastasis specimens, but was clearly detectable also in the normalliver counterpart. By contrast, ED-A-derived peptides gave strongsignals in the metastasis samples, but were completely undetectable(i.e., >100-fold lower signal) in normal liver.

Immunohistochemistry

The most striking discrimination between liver structures and metastaticneovasculature was observed for the ED-A and ED-B domains offibronectin. In both cases, a strong and specific staining of themetastatic blood vessels was observed, while normal liver and virtuallyall normal organs (exception made for the endometrium in theproliferative phase and some vessels of the ovaries) scored negative inthis immunohistochemical analysis (FIG. 3A). Importantly, ED-A was alsofound to be strongly expressed in the neo-vasculature of human lungmetastases and liver metastases (FIG. 4).

In Vivo Targeting

In order to test the usefulness of ED-A as a target for ligand-basedvascular targeting of metastases, an in vivo targeting experiment usingnear-infrared fluorescence imaging was performed. The parent anti-ED-AscFv antibody was labelled with Alexa Fluor 750 and injectedintravenously into F9 metastases-bearing mice. Near-infraredfluorescence imaging of the excised organs revealed a strikingaccumulation of the targeting agent in the metastatic lesions (FIG. 3B).

Selection of Anti-ED-A Antibodies BIAcore Analysis 1

The BIAcore analysis produced a graph for each anti-ED-A antibody whichwas analysed to deduce the affinity of an antibody for the antigen asfollows: The x axis of each graph corresponds to time and the y axiscorresponds to Resonance Units (a measure which indicates the bindingaffinity of the tested antibody for the antigen coated onto the BIAcorechip). Each graph showed 3 peaks and 1 dip which correspond to changesof buffer and are therefore irrelevant for the interpretation of theresults.

The ascending part of each graph represents the association phase. Thesteeper the curve in this part of the graph, the faster the associationof the antibody with the antigen. The descending part of each graphrepresents the dissociation phase of the antibody from the antigen. Theflatter the curve in this part of the graph is, the slower thedissociation of the antibody from the antigen.

Anti-ED-A antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 allshowed a flatter dissociation curve than the parent anti-ED-A antibodyfrom which they were derived, indicating that they bind ED-A, and hencealso A-FN, with a greater affinity than the parent anti-ED-A antibody.The graphs for antibodies E5, F1, F8 and H1 showed the flattestdissociation curves of all the anti-ED-A antibodies tested. Theassociation curves of antibodies H1, C5, D5, E5, C8, F8 and F1 wereflatter than that observed for the parent anti-ED-A antibody while theassociation curve observed for antibodies B2, B7, E8 and G9 was as steepas the association curve observed for the parent anti-ED-A antibody.However, as bacterial supernatants of IFTG-induced E. coli TG-1 cellswere used for the BIAcore analysis of antibodies H1, B2, C5, D5, E5, C8,F8, F1, B7, E8 and G9, the concentration of the tested antibody sampleswas unknown but most probably lower than the concentration of the parentanti-ED-A antibody sample used for comparison. Consequently, theassociation curve of antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8and G9 may be artificially low due to the low concentration of antibodyin the samples used for the BIAcore analysis. However, as concentrationdoes not significantly affect the dissociation of an antibody from itstarget antigen in BIAcore analysis, the flat dissociation curvesobserved for antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9show that these antibodies bind ED-A with at least an equal, andprobably a higher affinity, than the parent anti-ED-A antibody.Consequently, anti-ED-A antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7,E8 and G9 are extremely likely to give rise to the same or betterresults when used in the same in vivo and immunohistochemical studiesconducted using the parent anti-ED-A antibody as described elsewhereherein. The in vivo and immunohistochemical data obtained using theparent anti-ED-A antibody therefore provides evidence that anti-ED-Aantibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 and 09 may be used forthe treatment of tumour metastases.

BIAcore Analysis

The k_(on), k_(off) and KD values for each anti-ED-A antibody wereevaluated using the BIAevaluation software. The k_(on), k_(off) and KDvalues of the parent anti-ED-A antibody and anti-ED-A antibodies B2, C5,D5, C8, F8, B7 and G9 for antigen 11A12 are detailed in Table 3.Anti-ED-A antibodies B2, C5, D5, C8, F8, B7 and G9 all have a betterK_(D) values for antigen 11A12 than the parent anti-ED-A antibody fromwhich they were derived, indicating that they bind ED-A, and hence alsoA-FN, with a greater affinity than the parent anti-ED-A antibody.Consequently, anti-ED-A antibodies B2, C5, D5, C8, F8, B7 and G9 areextremely likely to give rise to the same or better results when used inthe same in vivo and immunohistochemical studies conducted using theparent anti-ED-A antibody as described elsewhere herein. The in vivo andimmunohistochemical data obtained using the parent anti-ED-A antibodytherefore provides evidence that anti-ED-A B2, C5, D5, C8, F8, B7 and G9may be used for the treatment of tumour metastases.

Biodistribution of F8 Diabody

The percentage (%) of the injected dose (ID) of I¹²⁵ labelled(iodinated) F8 diabody detected per gram (g) of mouse tissue was verysimilar for the liver, lung, spleen, heart, kidney, intestine, tail andblood and all, with the exception of the kidney, showed less than 2%ID/g (FIG. 8). In contrast, the F9 tumours contained on average aboutfour times more of the ID than any of the other mouse tissues analyzed(FIG. 8). This demonstrates that the F8 diabody was selectively targetedto the F9 mouse tumours. The percentage of the ID detected in the othertissues most likely represents background load of F8 diabody present inthe mice or non-specific labelling of the other mouse tissues. Asdescribed elsewhere herein, the biodistribution experiment was performedusing four mice and although the percentage of the ID detected per mousetissue varied (see error bars in FIG. 8) the percentage of F8 diabodydetected in the F9 tumours was consistently higher than in any of theother mouse tissues tested.

The biodistribution study was conducted on F9 primary tumours, and theresults indicate that the anti-ED-A antibodies in accordance with thepresent invention are selectively targeted to tumour tissue in vivo. Theresults provide further indication that the anti-ED-A antibodies of thepresent invention can be used to achieve the same or better results whenused in the same in vivo and immunohistochemical studies conducted usingthe parent anti-EDA antibody as described elsewhere herein.

Sequencing

Anti-ED-A antibodies H1, B2, C.5, D5, E.5, C8, F8, F1, E7, E8 and G9 areall scFv antibodies and were sequenced using conventional methods. Thenucleotide sequence of the anti-ED-A antibody H1 is shown in FIGS.6A-6C. The amino acid sequence of the anti-ED-A antibody H1 is shown inFIGS. 7A-7C.

Preferred nucleotide sequences encoding VH and/or VL of anti-ED-Aantibodies B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 are identical tonucleotide sequences encoding VH and/or VL of anti-ED-A antibody H1,except that the nucleotide sequences encoding the H1 CDR1s of the light(VL) and heavy (VH) chain are substituted with the nucleotide sequencesencoding the light (VL) and heavy (VH) chain CDR1s listed in Table 2 forthe respective antibody.

Some preferred nucleotide sequences encoding the VH and/or VL domains ofanti-ED-A F8 diabody are identical to the nucleotide sequences encodingVH and/or VL domains of anti-ED-A antibody H1, except that thenucleotide sequences encoding the H1 CDR1s of the light (VL) and heavy(VH) chain are substituted with the nucleotide sequences encoding thelight (VL) and heavy (VH) chain CDR's listed in Table 2 for anti-ED-Aantibody F8. A preferred nucleotide sequence encoding the linker linkingthe VH and VL domains of the anti-ED-A F8 diabody is gggtccagtggcggt(SEQ ID HO: 29).

Anti-ED-A antibodies B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 haveidentical amino acid sequences to anti-ED-A antibody H1, except that theamino acid sequences of the H1 CDR1s of the light (VL) and heavy (VH)chain are substituted with the amino acid sequences of the light (VL)and heavy (VH) chain CDR1s listed in Table 2 for the respectiveantibody. The amino acid sequences of the VH and VL domains of anti-ED-AF8 diabody are identical to the amino acid sequences of anti-ED-Aantibody H1, except that the amino acid sequences of the H1 CDR1s of thelight (VL) and heavy (VH) chain are substituted with the amino acidsequences of the light (VL) and heavy (VH) chain CDR1s listed in Table 2for anti-ED-A antibody F8, and the amino acid sequence of the linker inH1 is substituted with the linker amino acid sequence GSSGG (SEQ ID NO:28).

The amino acid sequence of the anti-ED-A antibody B2 VH domain (SEQ IDNO: 21) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 23 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody C5 VH domain (SEQ IDNO: 41) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 43 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody D5 VH domain (SEQ IDNO: 51) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 53 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody E5 VH domain (SEQ IDNO: 61) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 63 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody C8 VH domain (SEQ IDNO: 71) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 73 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody F8 VH domain (SEQ IDNO: 81) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 83 is substituted for theVH CDR1 of H1. The VH domains of the anti-ED-A F8 diabody have the sameamino acid sequence as VH domain of the anti-ED-A scFv antibody F8 (i.e.SEQ ID NO: 81).

The amino acid sequence of the anti-ED-A antibody F1 VH domain (SEQ IDNO: 91) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 93 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody B7 VH domain (SEQ IDNO: 101) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 103 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody E8 VH domain (SEQ IDNO: 111) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 113 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody G9 VH domain (SEQ IDNO: 31) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 33 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody B2 VL domain (SEQ IDNO: 22) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 26 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody B5 VL domain (SEQ IDNO: 42) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 46 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody D5 VL domain (SEQ IDNO: 52) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 56 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody E5 VL domain (SEQ IDNO: 62) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 66 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody C8 VL domain (SEQ IDNO: 72) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 76 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody F8 VL domain (SEQ IDNO: 82) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 86 is substituted for theVL CDR1 of H1. The VL domains of the anti-ED-A F8 diabody have the sameamino acid sequence as VL domain of the anti-ED-A antibody F8 (i.e. SEQID NO: 82).

The amino acid sequence of the anti-ED-A antibody F1 VL domain (SEQ IDNO: 92) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 96 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody B7 VL domain (SEQ IDNO: 102) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 106 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody E8 VL domain (SEQ IDNO: 112) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 116 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody G9 VL domain (SEQ IDNO: 32) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 36 is substituted for theVL CDR1 of H1.

Optionally, the amino acid at position 5 of the VH domain of anti-ED-Aantibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8, G9 may be a leucineresidue (L) rather than a valine residue (V) as shown in FIG. 7A. Inaddition, or alternatively, the amino acid at position 18 of the VLdomain of anti-ED-A antibodies H1, B2, C5, D5, E5, C5, F8, E1, B7, E8,G9 may be an arginine residue (R) rather than a lysine residue (K) asshown in FIG. 7C.

REFERENCES

All references cited anywhere in this specification, including thosecited anywhere above, are hereby incorporated by reference in theirentirety and for all purposes.

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TABLE 1 Fibronectin peptides identified in normal liver and/ormetastasis Seq. Position ¹Liver ¹Metastasis Peptide sequence Start EndTotal = 6) (Total = 8) HYQINQQWER 59 68 6 VGDTYERPK 109 117 4HALQSASAGSGSFTDVR 274 290 7 IGDQWDK 480 486 1 TFYQIGDSWEK 568 578 1WKEATIPGHLNSYTIK 654 669 2 EATIPGHLNSYTIK 656 669 1GLTPGVIYEGQLISIQQYGHR 670 690 7 WSRPQAPITGYR 830 841 2 3SDNVPPPTDLQFVELTDVK 903 921 3 VTIMWTPPDSVVSGYR 922 937 8 VEVLPVSLPGEHGQR938 952 8 NTFAEITGLSPGVTYLFK 958 975 7 VFAVHQGR 976 983 7 TVLVTWTPPR1011 1020 2 8 QYNVGPLASK 1040 1049 4 NLQPGSEYTVTLVAVK 1054 1069 6ATGVFTTLQPLR 1077 1088 1 8 LGVRPSQGGEAPR 1116 1128 7VVTPLSPPTNLHLEANPDTGVLTVSWER 1169 1196 3 STTPDITGYR 1197 1206 7VTWAPPPSIELTNLLVR 1375 1391 2 7 TGLDSPTGFDSSDITANSFTVHWVAPR 1446 1472 4APITGYIIR 1473 1481 1 8 HHAEHSVGRPR 1482 1492 1 EESPPLIGQQATVSDIPR 15251542 8 ITYGETGGNSPVQEFTVPGSK 1570 1590 2 8 SPVQEFTVPGSK 1579 1590 6STATINNIKPGADYTITLYAVTGR 1591 1614 5 GDSPASSKPVSINYK 1615 1629 4TEIDKPSQMQVTDVQDNSISVR 1630 1651 8 WLPSTSPVTGYR 1652 1663 7TASPDQTEMTIEGLQPTVEYVVSVYAQNR 1679 1707 7 ²NGESQPLVQTAVTTIPAPTNLK 17081819 3 ³NGESQPLVQTAVTNIDRPK 1708 1726 1 ³IAWESPQGQVSR 1740 1751 8³VTYSSPEDGIR 1754 1764 1 FSQVTPTSFTAQWIAPSVQLTGYR 1820 1843 1 5YEVSVYALK 1878 1886 2 TKTETITGFQVDAIPANGQTPVQR 1926 1949 1 2TETITGFQVDAIPANGQTPVQR 1928 1949 3 SYTITGLQPGTDYK 1957 1970 7IHLYTLNDNAR 1971 1981 7 SSPVIIDASTAIDAPSNLR 1982 2000 3 8FLTTTPNSLLVSWQAPR 2001 2017 4 5 ITGYIIK 2020 2026 5 YEKPGSPPR 2027 20356 ⁴PYLPNVDEEVQIGHVPR 2165 2181 7 GVTYNIIVEALQNQR 2255 2269 4 7RPGAAEPSPDGTTGHTYNQYTQR 2425 2447 2 ¹Numbers indicate in how many of the6 healthy in vivo biotinylated mice or the 8 metastases-bearing in vivobiotinylated mice the peptide was identified in the corresponding tissuesamples. All peptides are listed here which had been annotated by theMascot software to the fibronectin database entries P11276, Q3UHL6 orQ3TCF1. ²This pepetide covers a fibronectin sequence portion before ANDafter the ED-A domain, indicating the presence of an (EDA⁻)-fibronectinisoform. ³These peptides match to the sequence of the ED-A domain (Seq.positions 1721-1810). ⁴This peptide matches to the sequence of the IIICSstretch (Seq. positions 2082-2201).

TABLE 2 Nucleotide and amino acid sequences of the heavy chain (VH) andlight chain (VL) CDR1s of the anti-ED-A affinity matured antibodiesAntibody CDR1 (VH) CDR1 (VL) H1 CCG CGG AGG TCT GCG TGG P   R   R (SEQID NO: 3) S   A   W (SEQ ID NO: 6) B2 GCG GCT AAG GTG GCT TTT A   A   K(SEQ ID NO: 23) V   A   F (SEQ ID NO: 26) C5 CCG ATT ACT TTG CAT TTTP   I   T (SEQ ID NO: 43) L   H   F (SEQ ID NO: 46) D5 GTG ATG AAG AATGCT TTT V   M   K (SEQ ID NO: 53) N   A   F (SEQ ID NO: 56) E5 ACT GGTTCT CTT GCG CAT T   G   S (SEQ ID NO: 63) L   A   H (SEQ ID NO: 66) C8CTT CAG ACT CTT CCT TTT L   Q   T (SEQ ID NO: 73) L   P   F (SEQ ID NO:76) F8 CTG TTT ACG ATG CCG TTT L   F   T (SEQ ID NO: 83) M   P   F (SEQID NO: 86) F1 TAG GCG CGT GCG CCT TTT Q(Amber) A R (SEQ ID NO: 93)A   P   F (SEQ ID NO: 96) B7 CAT TTT GAT CTG GCT TTT H   F   D (SEQ IDNO: 103) L   A   F (SEQ ID NO: 106) E8 GAT ATG CAT TCG TCT TTT D   M   H(SEQ ID NO: 113) S   S   F (SEQ ID NO: 116) G9 CAT ATG CAG ACT GCT TTTH   M   Q (SEQ ID NO: 33) T   A   F (SEQ ID NO: 36)

TABLE 3 BIAcore evaluation data Antibody k_(on) (1/Ms) k_(off) (1/s)K_(D) (M) Parent anti-ED-A  2.5 × 10⁵ 0.02  ~1 × 10⁻⁷ antibody B2  3.8 ×10⁵ 7.54 × 10⁻³   ~2 × 10⁻⁸ C5 3.04 × 10⁵ 9.23 × 10⁻³   ~3 × 10⁻⁸ D54.53 × 10⁵ 7.6 × 10⁻³ ~1.7 × 10⁻⁸ C8  3.8 × 10⁵ 5.3 × 10⁻³ ~1.4 × 10⁻⁸F8 4.65 × 10⁵ 1.4 × 10⁻³ ~3.1 × 10⁻⁹ B7 2.67 × 10⁵ 4.5 × 10⁻³ ~1.68 ×10⁻⁸  G9  3.6 × 10⁵ 7.54 × 10⁻³  ~2.09 × 10⁻⁸ 

1-57. (canceled)
 58. An isolated antibody, or an antigen-bindingfragment thereof, that binds the Extra Domain-A (ED-A) of fibronectin,wherein the antibody comprises a heavy chain variable (VH) domain and alight chain variable (VL) domain, wherein the VH domain comprises theheavy chain CDR1, CDR2 and CDR3 amino acid sequences in SEQ ID NO: 81;and the VL domain comprises the light chain CDR1, CDR2 and CDR3 aminoacid sequences in SEQ ID NO:
 82. 59. The antibody or antigen-bindingfragment of claim 58, wherein at least one of the VH domain and the VLdomain comprises a human framework.
 60. The antibody or antigen-bindingfragment of claim 59, wherein the human VH domain framework is from ahuman germline DP47 gene and/or the human VL domain framework is from ahuman germline DPK22 gene.
 61. The antibody or antigen-binding fragmentof claim 58, wherein the VH domain comprises the amino acid sequence ofSEQ ID NO: 81; or the amino acid sequence of SEQ ID NO: 81 wherein theamino acid at position 5 of SEQ ID NO: 81 is a leucine residue (L)rather than a valine residue (V).
 62. The antibody or antigen-bindingfragment of claim 58, wherein the VL domain comprises: (i) the aminoacid sequence of SEQ ID NO: 82; (ii) the amino acid sequence of SEQ IDNO: 82 wherein the amino acid at position 18 of SEQ ID NO: 82 is anarginine residue (R) rather than a lysine residue (K); (iii) amino acidresidues 1-108 of SEQ ID NO: 82; or (iv) amino acid residues 1-108 ofSEQ ID NO: 82 wherein the amino acid at position 18 of SEQ ID NO: 82 isan arginine residue (R) rather than a lysine residue (K).
 63. Theantibody or antigen-binding fragment of claim 58, wherein the VH domaincomprises the amino acid sequence of SEQ ID NO: 81 wherein the aminoacid at position 5 of SEQ ID NO: 81 is a leucine residue (L) rather thana valine residue (V); and the VL domain comprises amino acid residues1-108 of SEQ ID NO: 82 wherein the amino acid at position 18 of SEQ IDNO: 82 is an arginine residue (R) rather than a lysine residue (K). 64.The antibody or antigen-binding fragment of claim 63, wherein theantibody or antigen-binding fragment is conjugated to a detectable labelor a functional label.
 65. The antibody or antigen-binding fragment ofclaim 63, wherein the antibody or antigen-binding fragment is conjugatedto a biocidal or cytotoxic molecule, or to a radioisotope.
 66. Theantigen-binding fragment of claim 58, comprising a single chain Fv(scFv).
 67. The antigen-binding fragment of claim 66, wherein the VHdomain is conjugated to the VL domain via a peptide linker.
 68. Theantigen-binding fragment of claim 67, wherein the peptide linkercomprises 5 to 25 amino acids.
 69. The antigen-binding fragment of claim68, wherein the peptide linker has 5 amino acids.
 70. Theantigen-binding fragment of claim 66, wherein the VH domain of the ScFvcomprises the amino acid sequence of SEQ ID NO: 81 wherein the aminoacid at position 5 of SEQ ID NO: 81 is a leucine residue (L) rather thana valine residue (V); and the VL domain of the ScFv comprises amino acidresidues 1-108 of SEQ ID NO: 82 wherein the amino acid at position 18 ofSEQ ID NO: 82 is an arginine residue (R) rather than a lysine residue(K); wherein the VH domain is conjugated to the VL domain via a peptidelinker; and wherein the antigen-binding fragment is conjugated to afunctional label.
 71. The antigen-binding fragment of claim 66, whereinthe VH domain of the ScFv comprises the amino acid sequence of SEQ IDNO: 81 wherein the amino acid at position 5 of SEQ ID NO: 81 is aleucine residue (L) rather than a valine residue (V); and the VL domainof the ScFv comprises amino acid residues 1-108 of SEQ ID NO: 82 whereinthe amino acid at position 18 of SEQ ID NO: 82 is an arginine residue(R) rather than a lysine residue (K); wherein the VH domain isconjugated to the VL domain via a peptide linker; and wherein theantigen-binding fragment is conjugated to a biocidal or cytotoxicmolecule, or to a radioisotope.
 72. The antigen-binding fragment ofclaim 58, wherein the antigen-binding fragment is a diabody thatcomprises a polypeptide comprising the VH and VL domains, wherein the VHdomain is conjugated to the VL domain via a peptide linker.
 73. Theantigen-binding fragment of claim 72, wherein the peptide linker has 1to 5 amino acids.
 74. The antigen-binding fragment of claim 73, whereinthe peptide linker has 5 amino acids.
 75. The antigen-binding fragmentof claim 72, wherein the VH domain of the polypeptide comprises theamino acid sequence of SEQ ID NO: 81 wherein the amino acid at position5 of SEQ ID NO: 81 is a leucine residue (L) rather than a valine residue(V); and the VL domain of the polypeptide comprises amino acid residues1-108 of SEQ ID NO: 82 wherein the amino acid at position 18 of SEQ IDNO: 82 is an arginine residue (R) rather than a lysine residue (K); andwherein the VH domain is conjugated to the VL domain via a peptidelinker having 5 amino acids.
 76. The antigen-binding fragment of claim75, wherein the antigen-binding fragment is conjugated to a functionallabel.
 77. The antigen-binding fragment of claim 75, wherein theantigen-binding fragment is conjugated to a biocidal or cytotoxicmolecule, or to a radioisotope.
 78. A pharmaceutical compositioncomprising the antibody or antigen-binding fragment of claim 58 and apharmaceutically acceptable carrier.